Monoclonal antibody capable of binding to heparin-binding epidermal growth factor-like growth factor

ABSTRACT

Medicaments for treating diseases related to HB-EGF escalation are in demand. The present invention provides a monoclonal antibody or an antibody fragment thereof which binds to a cell membrane-bound HB-EGF, a membrane type HB-EGF and a secretory HB-EGF.

TECHNICAL FIELD

The present invention relates to a monoclonal antibody or an antibodyfragment thereof which binds to a cell membrane-bound heparin bindingepidermal growth factor-like growth factor (hereinafter referred to as“HB-EGF”), a membrane type HB-EGF and a secretory HB-EGF.

BACKGROUND ART

HB-EGF was isolated and purified by Higashiyama et al. in 1992 from aculture supernatant of a macrophage-differentiated human macrophage-likecell line U-937 (Non-patent document 1). HB-EGF holds 6 cysteineresidues in common preserved in the epidermal growth factor (EGF) familyand belongs to the EGF family, and is synthesized as a type I membraneprotein similar to the case of other proteins belonging to the EGFfamily (Non-patent documents 1 and 2). The membrane type HB-EGF isconverted into a secretory HB-EGF of 14 to 22 kilo daltons (hereinafterreferred to as “kDa”) by a metalloprotease activated by variousphysiological stimuli such as stress due to heat or osmotic pressure, agrowth factor, a cytokine and lysophosphatidic acid (LPA) which is a Gprotein coupled receptor (GPCR) agonist (Non-patent documents 1 to 3).The secretory HG-EGF binds to an EGF receptor (EGFR/ErbB 1) (Non-patentdocument 1), ErbB4 (Non-patent document 4) and N-arginine dibasicconvertase (Non-patent document 5), and has the growth accelerationactivity for fibroblasts and smooth muscle cells (Non-patent document1), keratinocyte (Non-patent document 6), hepatocyte (Non-patentdocument 7) and mesangial cell (Non-patent document 8). In addition, itis also known that HB-EGF is related to organogenesis of, for example,cardiac valve (Non-patent documents 28, 29 and 31), healing of wound(Non-patent documents 9 and 10), hyperplasia of smooth muscle cellcaused in atherosclerosis (Non-patent document 11), re-stricture(Non-patent documents 12 and 13), pulmonary hypertension (Non-patentdocument 14), hepatic regeneration (Non-patent document 15), cerebraldisorder (Non-patent document 16) and cancer (Non-patent documents 28 to35).

On the other hand, it has been reported that a considerable amount ofmembrane type HB-EGF is expressed on the cell surface without beingdigested into its secretory (Non-patent document 17). It is known thatthe membrane type HB-EGF forms a complex on the cell surface with CD9 orthe like tetra tetraspanin or integrin α3β1, and it has been reportedalso that it interacts as a juxtacrine growth factor with adjacent cells(Non-patent documents 17 to 22). In addition, Naglich et al. havereported that the membrane type HB-EGF functions as receptor ofdiphtheria toxin and is related to the internalization of diphtheriatoxin into cells (Non-patent document 23).

When Mekada et al. have analyzed physiological functions of HB-EGF bypreparing HB-EGF knockout (KO) mice, the HB-EGF KO mice showed dilationof ventricle, lowering of cardiac function and a symptom of cardiacvalve hypertrophy and more than half of the animals died in several daysafter birth. This fact shows that HB-EGF is a protein essential for thedevelopment and functional maintenance of the heart (Non-patent document24).

Next, Mekada et al. have prepared two genes for an HB-EGF which becameunable to be converted into secretory due to introduction of a mutationinto a protease digestion site (hereinafter referred to as “HB^(uc)”)and an HB-EGF which lacks a transmembrane region, is secreted and issecreted independently of protease digestion (hereinafter referred to as“HB^(Δtm)”). By preparing transgenic mice which express respectiveHB-EGF mutants, physiological functions of membrane type and secretoryHB-EGFs were analyzed (Non-patent document 25). As a result, since theHB^(uc) expressing mice showed symptoms similar to those of the HB-EGFKO mice, it was considered that the secretory HB-EGF is functioning asthe active type protein. Most of the HB^(Δtm) expressing mice diedbefore the neonatal stage or at the neonatal stage. In addition,hyperplasia of keratinocyte and ventricular hypertrophy from theneonatal stage were found in HB^(Δtm/+) expressing mice in which amutation was introduced into only one of the alleles. These symptomswere phenotypes directly opposite to those of the HB-EGF KO mice andHB^(uc) mice. CRM197 known as a mutant of diphtheria toxin (Non-patentdocument 26) specifically inhibits cell growth acceleration activity ofHB-EGF and does not permeate cell membrane. Since this CRM197 inhibitedhyperplasia and ventricular hypertrophy as phenotypes of the HB^(Δtm)expressing mice, it is considered that the HB^(Δtm) formed in theHB^(Δtm) expression mice does not act by binding to its intracellularreceptor before its secretion, but acts by binding to the receptor onthe cell surface after secreted extracellularly. Accordingly, thequantitative balance between membrane type HB-EGF and secretory HB-EGFin the living body is essential for the maintenance of normalphysiological functions, and it is considered that the process forconverting from membrane type into secretory of HB-EGF is controlled inthe living body.

Higashiyama et al. have found that secretory HB-EGF protein in the heartis increased in the heart of a mouse in which cardiac hypertrophy wasinduced by constricting the thoracic aorta. It has been reported thatwhen a low molecular weight compound capable of inhibiting a proteasewhich converts membrane type HB-EGF into secretory is administered tothis mouse, cardiac hypertrophy is suppressed as a result of suppressingconversion of the membrane type HB-EGF into secretory in the heart(Non-patent document 27).

It has been reported so far that HB-EGF is expressed at a high level invarious cancers such as breast cancer, liver cancer, pancreas cancer andbladder cancer, in comparison with normal tissues (Non-patent documents28 to 31). Also, it has been recently found that HB-EGF is an importantfactor for the proliferation of cancer (Non-patent documents 32 and 33).Mekada et al. have found that a significant tumor growth inhibitoryeffect is recognized when small interference RNA (siRNA) of HB-EGF isintroduced into a cancer cell line, or CRM197 is administered to a mouseinto which the cancer cell line was transplanted, in a model system inwhich a human ovarian cancer cell line is transplanted into a nudemouse. Also, Higashiyama et al. have found that cell growth, colonyforming ability, vascular endothelial growth factor (VEGF) expressionand expression of cyclin D1 and the like are increased in vitro in abladder cancer cell line into which the HB-EGF gene was transferred. Inaddition, it was reported that increase of tumorigenicity and increaseof tumor angiogenesis are found also in vivo. Such a growth stimulationactivity was found only when the membrane type HB-EGF gene or secretoryHB-EGF gene was expressed, but was nor found when a protease-resistantmembrane type HB-EGF gene was forcedly expressed. Accordingly, apossibility was suggested that the secretory HB-EGF is an importantfactor which is related to the tumor growth of ovarian cancer andbladder cancer. Regarding the expression of HB-EGF in clinical patients,Mekada et al. have analyzed expression quantity of HB-EGF mRNA andconcentration of secretory HB-EGF in the tumor tissues and ascites ofovarian cancer patients, and reported that only HB-EGF among the EGFfamily is expressed (Non-patent document 32). In addition, Miyamoto etal. have reported that prognosis is poorer in ovarian cancer patients inwhich HB-EGF mRNA of the tumor is highly expressed, than low expressionpatients (Non-patent document 34). The above results show that at leastin the ovarian cancer, the secretory HB-EGF produced by the cancer isrelated to the cancer growth by the autocrine or paracrine mechanism(Non-patent document 35). As antibodies which bind to secretory HB-EGFand inhibit its activity, some polyclonal antibodies and one monoclonalantibody (all manufactured by R & D) are known. It has been reportedthat an anti-HB-EGF goat polyclonal antibody (manufactured by R & D)binds to the cell surface membrane type HB-EGF expressed in COS-7 cell(Non-patent document 3). It is broadly known that when a membraneprotein is present on the surface of a cell such as cancer, a monoclonalantibody which binds to such a protein could become a therapeutic agentwhich inhibits growth of the cell (Non-patent document 36).

It is known that generally, when a non-human animal antibody such as amouse antibody is administered to human, it is recognized as a foreignsubstance so that a human antibody for mouse antibody [human anti mouseantibody (HAMA)] is induced in the human body. It is known that HAMAreacts with the administered mouse antibody to thereby induce sideeffects (Non-patent Documents 37 to 40), increases elimination of themouse antibody from the body (Non-patent Documents 38, 41, and 42) anddecreases therapeutic effect of the mouse antibody (Non-patent Documents43 and 44).

In order to solve these problems, attempts have been made to preparerecombinant antibodies such as a human chimeric antibody or a humanizedantibody from a non-human antibody using gene recombination techniques.

A human chimeric antibody and a humanized antibody have variousadvantages in administration to human in comparison with a non-humanantibody such as a mouse antibody in a clinical application. Forexample, it has been reported that its immunogenicity was decreased andits half-life in blood was prolonged in a test using monkey, incomparison with a mouse antibody (Non-patent Documents 45 and 46). Thatis, since the human chimeric antibody and the humanized antibody causefewer side effects in human than non-human antibodies, it is expectedthat its therapeutic effect is sustained for a prolonged time.

Also, since a human chimeric antibody and a humanized antibody areprepared using gene recombination techniques, it can be prepared asvarious forms of molecules. For example, when γ1 subclass is used as aheavy chain (hereinafter referred to as “H chain”) constant region(hereinafter referred to as “C region”) of a human antibody (H chain Cregion is referred to as “CH”), a human chimeric antibody and ahumanized antibody having high effector functions such asantibody-dependent cellular cytotoxicity (hereinafter referred to as“ADCC activity”) can be prepared (Non-patent Document 14), andprolongation of its half-life in blood in comparison with mouseantibodies can be expected (Non-patent Document 46). Particularly, inthe case of treatment for decreasing cells expressing a membrane typeHB-EGF or cells having a cell membrane in which a secretory HB-EGF isbound to the surface thereof, the degree of cytotoxic activities such ascomplement-dependent cytotoxicity (hereinafter referred to as “CDCactivity”) via the Fc region (the region after the antibody heavy chainhinge region) of an antibody and ADCC activity is important for itstherapeutic efficacy. In the treatment of human, a human chimericantibody, a humanized antibody or a human antibody is preferably usedfor exerting the cytotoxic activities (Non-patent Documents 47 and 48).

In addition, with recent advance in protein engineering and geneticengineering, human chimeric antibody or the humanized antibody can alsobe prepared as an antibody fragment having a low molecular weight, suchas Fab, Fab′, F(ab′)₂, a single chain antibody (hereinafter referred toas “scFv”) (Non-patent Document 49), a dimerized V region fragment(hereinafter referred to as “diabody”) (Non-patent Document 51), adisulfide stabilized V region fragment (hereinafter referred to as“dsFv”) (Non-patent Document 52), or a peptide comprising acomplementarity determining region (hereinafter referred to as “CDR”)(Non-patent Document 50), and these antibody fragments have moreexcellent migrating ability to target tissues than full antibodymolecules (Non-patent Document 53).

The above facts show that a human chimeric antibody or a humanizedantibody is preferable to a non-human animal antibody such as a mouseantibody as an antibody to apply to human in a clinical setting.

Non-patent document 1: Science, Vol. 251, 936, 1991Non-patent document 2: J. Biol. Chem. 267 (1992) 6205-6212Non-patent document 3: Nature, Vol. 402, 884, 1999Non-patent document 4: EMBO J. 16 (1997) 1268-1278Non-patent document 5: EMBO J. 20 (2001) 3342-3350Non-patent document 6: J. Biol. Chem. 269 (1994) 20060-20066Non-patent document 7: Biochem Biophys. Res. Commun. 198 (1994) 25-31Non-patent document 8: J. Pathol. 189 (1999) 431-438Non-patent document 9: Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 3889-3893Non-patent document 10: J. Cell Biol. 151 (2000) 209-219Non-patent document 11: J. Clin. Invest., 95, 404, 1995Non-patent document 12: Arterioscler. Thromb. Vasc. Biol. 16 (1996)1524-1531Non-patent document 13: J Biol. Chem. 277 (2002) 37487-37491Non-patent document 14: Am. J. Pathol. 143 (1993) 784-793Non-patent document 15: Hepatology 22 (1995) 1584-1590Non-patent document 16: Brain Res. 827 (1999) 130-138Non-patent document 17: Biochem. Biophys. Acta., Vol. 1333, F179, 1997Non-patent document 18: J. Cell Biol. 128 (1995) 929-938Non-patent document 19: J. Cell Biol. 129 (1995) 1691-1705Non-patent document 20: Cytokine Growth Factor Rev., Vol. 11, 335, 2000Non-patent document 21: Int. J. Cancer, Vol. 98, 505, 2002Non-patent document 22: J. Histochem. Cytochem., Vol. 49, 439, 2001Non-patent document 23: Cell, Vol. 69, 1051, 1992Non-patent document 24: PNAS, Vol. 100, 3221, 2003Non-patent document 25: J. of Cell Biology, Vol. 163, 469, 2003Non-patent document 26: J. Biol. Chem., Vol. 270, 1015, 1995Non-patent document 27: Nat. Med., Vol. 8, 35, 2002Non-patent document 28: Breast Cancer Res. Treat., Vol. 67, 81, 2001Non-patent document 29: Oncol. Rep., Vol. 8, 903, 2001Non-patent document 30: Biochem. Biophys. Res. Commun., Vol. 202, 1705,1994Non-patent document 31: Cancer Res., Vol. 61, 6227, 2001Non-patent document 32: Cancer Res., Vol. 64, 5720, 2004Non-patent document 33: Cancer Res., Vol. 64, 5283, 2004Non-patent document 34: Clin. Cancer Res., Vol. 11, 4783, 2005Non-patent document 35: Clin. Cancer Res., Vol. 11, 4639, 2005Non-patent document 36: Nat. Rev. Drug. Discov., Vol. 2, 52-62, 2003Non-patent Document 37: J. Clin. Oncol., 2,881 (1984)

Non-patent Document 38: Blood, 65,1349 (1985)

Non-patent Document 39: J. Natl. Cancer Inst., 80,932 (1988)Non-patent Document 40: Proc. Natl. Acad Sci. USA, 82, 1242 (1985)Non-patent Document 41: J. Nucl. Med., 26, 1011 (1985)Non-patent Document 42: J. Natl. Cancer Inst., 80, 937 (1988)

Non-patent Document 43: J. Immunol., 135, 1530 (1985) Non-patentDocument 44: Cancer Res., 46, 6489 (1986) Non-patent Document 45: CancerRes., 56, 1118 (1996) Non-patent Document 46: Immunol., 85, 668 (1995)Non-patent Document 47: J. Immunol., 144, 1382 (1990) Non-patentDocument 48: Nature, 322, 323 (1988) Non-patent Document 49: Science,242, 423 (1988) Non-patent Document 50: Nature Biotechnol., 15, 629(1997) Non-patent Document 51: Molecular Immunol., 32, 249 (1995)

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Non-patent Document 53: Cancer Res., 52, 3402 (1992) DISCLOSURE OF THEINVENTION Problems to be Solved by the Invention

Medicaments for treating diseases relating to HB-EGF are in demand.

Means to Solve the Problems

The present invention relates to the following (1) to (28):

(1) A monoclonal antibody or an antibody fragment thereof which binds toa cell membrane-bound heparin binding epidermal growth factor-likegrowth factor, a membrane type HB-EGF and a secretory HB-EGF.(2) The monoclonal antibody or the antibody fragment thereof accordingto (1), which binds to epidermal growth factor-like domain (EGF-likedomain) of the cell membrane-bound HB-EGF, the membrane type HB-EGF andthe secretory HB-EGF.(3) The monoclonal antibody or the antibody fragment thereof accordingto (1) or (2), which inhibits binding of the secretory HB-EGF and anHB-EGF receptor.(4) The monoclonal antibody or the antibody fragment thereof accordingto any one of (1) to (3), which has neutralizing activity for thesecretory HB-EGF.(5) The monoclonal antibody or the antibody thereof according to any oneof (1) to (4), which binds to a binding region of the secretory HB-EGFand an HB-EGF receptor or diphtheria toxin.(6) The monoclonal antibody or the antibody thereof according to any oneof (1) to (5), which binds to an epitope comprising at least one ofamino acids at positions 133, 135 and 147 in the amino acid sequencerepresented by SEQ ID NO:2.(7) The monoclonal antibody or the antibody fragment thereof accordingto (6), which binds to an epitope comprising amino acids at positions133, 135 and 147 in the amino acid sequence represented by SEQ ID NO:2.(8) The monoclonal antibody or the antibody fragment thereof accordingto any one of (1) to (5), which binds to an epitope comprising the aminoacid at position 141 in the amino acid sequence represented by SEQ IDNO:2.(9) The monoclonal antibody or the antibody fragment thereof accordingto any one of (1) to (3), (5) and (8), which binds to an epitope towhich a monoclonal antibody produced by hybridoma KM3579 (FERM BP-10491)binds.(10) The monoclonal antibody or the antibody fragment thereof accordingto any one of (1) to (7), which binds to an epitope to which amonoclonal antibody produced by hybridoma KM3567 (FERM BP-10573) binds.(11) The monoclonal antibody or the antibody fragment thereof accordingto any one of (1) to (7), which binds to an epitope to which amonoclonal antibody produced by hybridoma KM3566 (FERM BP-10490) binds.(12) The antibody or the antibody fragment thereof according to any oneof (1) to (11), wherein the monoclonal antibody is a recombinantantibody.(13) The antibody or the antibody fragment thereof according to (12),wherein the recombinant antibody is selected from a human chimericantibody, a humanized antibody and a human antibody.(14) The monoclonal antibody or the antibody fragment thereof accordingto (13), wherein CDR (complementarity determining region, hereinafterreferred to “CDR”) 1, CDR2 and CDR3 of a heavy chain variable region(hereinafter referred to “VH”) of an antibody comprise amino acidsequences represented by SEQ ID NOs:12, 13 and 14, respectively, andCDR1, CDR2 and CDR3 of a light chain variable region (hereinafterreferred to as “VL”) of an antibody comprise amino acid sequencerepresented by SEQ ID NOs:15, 16 and 17, respectively.(15) The human chimeric antibody or the antibody fragment thereofaccording to (13), wherein VH of the human chimeric antibody comprisesthe amino acid sequence represented by SEQ ID NO:9, and VL of the humanchimeric antibody comprises the amino acid sequence represented by SEQID NO:11.(16) The humanized antibody or the antibody fragment thereof accordingto (13), wherein VH of the humanized antibody comprises the amino acidsequence represented by SEQ ID NO:22 or an amino acid sequence in whichat least one modification selected from substitutions of Ala at position9 with Thr, Val at position 20 with Leu, Thr at position 30 with Arg,Arg at position 38 with Lys, Pro at position 41 with Thr, Met atposition 48 with Ile, Arg at position 67 with Lys, Val at position 68with Ala, Ile at position 70 with Leu, Tyr at position 95 with Phe, andVal at position 118 with Leu is introduced in the amino acid sequencerepresented by SEQ ID NO:22; and wherein VL of the humanized antibodycomprises the amino acid sequence represented by SEQ ID NO:23 or anamino acid sequence in which at least one modification selected fromsubstitutions of Leu at position 15 with Val, Ala at position 19 withVal, Ile at position 21 with Met, Pro at position 49 with Ser, and Leuat position 84 with Val is introduced in the amino acid sequencerepresented by SEQ ID NO:23.(17) The humanized antibody or the antibody fragment thereof accordingto (13), wherein VH of the humanized antibody comprises an amino acidsequence in which at least one modification among amino acidmodifications selected from substitutions of Val at position 20 withLeu, Thr at position 30 with Arg, Met at position 48 with Ile, Val atposition 68 with Ala, Ile at position 70 with Leu, Tyr at position 95with Phe, and Val at position 118 with Leu is introduced in the aminoacid sequence represented by SEQ ID NO:22, and wherein VL of thehumanized antibody comprises an amino acid sequence in which at leastone modification selected from substitutions of Leu at position 15 withVal, Ala at position 19 with Val, Ile at position 21 with Met, Pro atposition 49 with Ser, and Leu at position 84 with Val is introduced inthe amino acid sequence represented by SEQ ID NO:23.(18) The humanized antibody or the antibody fragment thereof accordingto (13), wherein VH of the humanized antibody comprises the amino acidsequence represented by SEQ ID NO:22, and VL of the humanized antibodycomprises the amino acid sequence represented by SEQ ID NO:43.(19) The humanized antibody or the antibody fragment thereof accordingto (13), wherein VH of the humanized antibody comprises the amino acidsequence represented by SEQ ID NO:42, and VL of the humanized antibodycomprises the amino acid sequence represented by SEQ ID NO:23.(20) The humanized antibody or the antibody fragment thereof accordingto (13), wherein VH of the humanized antibody comprises the amino acidsequence represented by SEQ ID NO:42, and VL of the humanized antibodycomprises the amino acid sequence represented by SEQ ID NO:43.(21) The antibody fragment according to any one of (1) to (20), which isselected from Fab, Fab′, F(ab′)₂, a single chain antibody (scFv), adimerized V region (diabody), a disulfide stabilized V region (dsFv),and a peptide comprising six CDRs.(22) A DNA encoding the antibody or the antibody fragment thereofaccording to any one of (1) to (21).(23) A recombinant vector comprising the DNA according to (22).(24) A transformant obtainable by introducing the recombinant vectoraccording to (23) into a host cell.(25) A process for producing the antibody or the antibody fragmentthereof according to any one of (1) to (21), which comprises culturingthe transformant according to (24) in a medium to form and accumulatethe antibody or the antibody fragment thereof according to any one of(1) to (21) in the culture, and recovering the antibody or the antibodyfragment from the culture.(26) A pharmaceutical composition comprising the antibody or theantibody fragment thereof according to any one of (1) to (21) as activeingredient.(27) An agent for treating a disease relating to HB-EGF, comprising theantibody or the antibody fragment thereof according to any one of (1) to(21) as an active ingredient.(28) The agent according to (27), wherein the disease relating to HB-EGFis cancer.

EFFECT OF THE INVENTION

The present invention provides a monoclonal antibody or an antibodyfragment thereof which binds to a cell membrane-bound heparin bindingepidermal growth factor-like growth factor, a membrane type HB-EGF and asecretory HB-EGF.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A It shows the reactivity of various anti-HB-EGF monoclonalantibodies by binding ELISA. The upper graph shows the result of thebinding ELISA to human HB-EGF and the lower graph shows the result ofthe binding ELISA to bovine serum albumin (BSA) as a negative control.The abscissa shows the concentration of each antibody, and the ordinateshows the binding activity of each antibody. ⋄ shows monoclonal antibodyKM511, ▪ shows monoclonal antibody KM3566, Δ shows monoclonal antibodyKM3567, ▴ shows monoclonal antibody KM3579, and ∘ shows monoclonalantibody MAB259.

FIG. 1B It shows the HB-EGF-EGFR binding inhibition activity of theanti-HB-EGF monoclonal antibodies KM3566, KM3567, KM3579 and MAB259. Theabscissa shows the concentration of each antibody, and the ordinateshows the binding of biotin-labeled HB-EGF, shown by fluorescenceintensity. The sidewise solid line shows fluorescence intensity at thetime when biotin-labeled HB-EGF was added and when the antibody was notadded, and the sidewise dotted line shows fluorescence intensity at thetime when biotin-labeled HB-EGF was not added and when the antibody wasnot added. Δ shows monoclonal antibody KM3566, x shows monoclonalantibody KM3567,  shows monoclonal antibody KM3579, and ▪ showsmonoclonal antibody MAB259.

FIG. 2A It shows the HB-EGF neutralization activity of variousanti-HB-EGF monoclonal antibodies. The abscissa shows the concentrationof each antibody, and the ordinate shows the growth inhibition ratio(%). ⋄ shows monoclonal antibody KM511, ▪ shows monoclonal antibodyKM3566, ▴ shows monoclonal antibody KM3579, and ∘ shows monoclonalantibody MAB259, respectively.

FIG. 2B It shows the HB-EGF neutralization activity of variousanti-HB-EGF monoclonal antibodies. The abscissa shows the concentrationof each antibody, and the ordinate shows the cell growth. HB-EGF(+)shows the cell growth at the time when HB-EGF was added and when theantibody was not added, and HB-EGF(−) shows the cell growth at the timewhen HB-EGF was not added and the antibody was not added. □ showsmonoclonal antibody MAB259, ▪ shows monoclonal antibody KM3567 and ▴shows monoclonal antibody KM3566.

FIG. 3 It shows the reactivity of various anti-HB-EGF monoclonalantibodies by FCM analysis. The abscissa shows the concentration of eachantibody, and the ordinate shows the mean fluorescence intensity. xshows monoclonal antibody KM511, Δ shows monoclonal antibody KM3566, □shows monoclonal antibody KM3579, and ∘ shows monoclonal antibodyMAB259. The dushed line shows the mean fluorescence intensity, MFIvalue, of a negative control without an antibody (at the time when theanti-HB-EGF monoclonal antibody was added and the FITC-labeled goatanti-mouse IgG+IgM (H+L) polyclonal antibody was not added).

FIG. 4 It shows the reactivity of various anti-HB-EGF monoclonalantibodies for MDA-MB-231 cell by FCM analysis. In each histogram, theleft peak shows negative control antibody KM511, and the right peakshows each anti-HB-EGF antibody. (a), (b), (c) and (d) show MAB529,KM3566, KM3567 and KM3579, respectively.

FIG. 5 It shows construction steps of an anti-HB-EGF chimeric antibodyexpression vector pKANTEX3566.

FIG. 6 It shows the SDS-PAGE (using 5 to 20% gradient gel)electrophoresis pattern of purified anti-HB-EGF chimeric antibodyKM3966. Lane 1 shows a molecular weight marker, lane 2 and lane 3 showthe anti-HB-EGF chimeric antibody KM3966 under reducing conditions andunder non-reducing conditions, respectively.

FIG. 7 It shows the reactivity of anti-HB-EGF chimeric antibody KM3966for a human solid carcinoma cell line by flow cytometry. In the drawing,the ordinate shows the number of cells, and the abscissa shows thefluorescence intensity.

FIG. 8 It shows the reactivity of anti-HB-EGF chimeric antibody KM3966for a recombinant HB-EGF-treated human solid carcinoma cell line by flowcytometry. In the drawing, the ordinate shows the number of cells, andthe abscissa shows the fluorescence intensity.

FIG. 9 It shows the neutralization activity of anti-HB-EGF chimericantibody KM3966 for human HB-EGF. In the drawing, the ordinate shows theabsorbance value at OD 450 nm which represents the number of viablecells, and the abscissa antibody shows the concentration. ▪ shows thenegative control antibody human IgG, and □ shows KM3966. HB-EGF (−)shows no addition of HB-EGF, and HB-EGF (+) shows addition of HB-EGF.

FIG. 10 It shows the antibody-dependent cellular cytotoxicity (ADCCactivity) of anti-HB-EGF chimeric antibody KM3966 for a human solidcarcinoma cell line. In the drawing, the ordinate shows the cytotoxicityratio (%), and the abscissa shows the antibody concentration ofanti-HB-EGF chimeric antibody KM3966. The sidewise straight line showsthe cytotoxicity at the time when the antibody was not added.

FIG. 11 It shows the antitumor activity of anti-HB-EGF chimeric antibodyKM3966 in an early cancer model. In the drawing, the ordinate shows thetumor volume, and the abscissa shows the number of days after cancercell transplantation.  shows PBS the administration group, and ∘ showsthe KM3966 10 mg/kg administration group. The bar shows the standarddeviation.

FIG. 12 It shows the antitumor activity of anti-HB-EGF chimeric antibodyKM3966 in an advanced cancer model. In the drawing, the ordinate showsthe tumor volume, and the abscissa shows the number of days after cancercell transplantation.  shows the PBS administration group, and ∘ showsthe KM3966 10 mg/kg administration group. The bar shows the standarddeviation.

FIG. 13 It shows the reactivity of anti-HB-EGF mouse antibody KM3566 fora human blood cancer cell line by flow cytometry. In the drawing, theordinate shows the number of cells, and the abscissa shows thefluorescence intensity. “A” shows the acute myelogenous leukemia cellline, and “B” shows the T cell leukemia cell line.

FIG. 14 It shows the antibody-dependent cellular cytotoxicity (ADCCactivity) of anti-HB-EGF chimeric antibody KM3966 for a human bloodcancer cell line. In the drawing, the ordinate shows the cytotoxicityratio (%), and the abscissa shows the antibody concentration ofanti-HB-EGF chimeric antibody KM3966. The sidewise straight line showsthe cytotoxicity at the time when the antibody was not added.

FIG. 15 It shows the reactivity of anti-HB-EGF antibodies to a humanovarian cancer cell lines MCAS. In the figure, the ordinate shows thenumber of cells, and the abscissa shows fluorescence intensity.

FIG. 16 It shows the neutralizing activity of anti-HB-EGF antibodies fora human gastric cancer cell line MKN-28. In the figure, the ordinateshows the cell proliferation, and the abscissa shows the proteinconcentration of added recombinant human HB-HGF and added anti-HB-EGFantibodies.

FIG. 17 It shows results in which the antibody-dependent cellularcytotoxicity (ADCC activity) of anti-HB-EGF humanized antibodies for ahematological cancer cell line was measured. The ordinate showscytotoxic activity (%), and the abscissa shows antibody concentration.The solid line remained steady shows cytotoxic activity at the time ofthe antibody non-addition.

FIG. 18 It shows the reactivity of anti-HB-EGF monoclonal antibodiesKM3566 and KM3579 and chimeric antibody KM3966 for mutant HB-EGFexpression cells. In the drawing, the ordinate shows the reactivity (%)of each antibody, and the abscissa shows kinds of mutant HB-EGF.

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, the membrane type HB-EGF is HB-EGF which bindsto a cell membrane through a cell membrane-spanning domain and consistsof a signal sequence, a pro-region, a heparin-binding domain, anEGF-like domain, a juxtamembrane domain and a cytoplasmic domain.Specifically, it includes a polypeptide comprising the amino acidsequence represented by SEQ ID NO:2. Also, in the present invention, thesecretory HB-EGF is an extracellular domain comprising an EGF-likedomain in which the membrane-binding region of the membrane type HB-EGFis cleaved by a protease or the like. Specifically, it includes apolypeptide comprising the amino acid sequence represented by SEQ IDNO:3. The cell membrane-bound HB-EGF is HB-EGF in which the secretoryHB-EGF is bound to the surface of a cell membrane by its haparin-bindingactivity, electrostatically binding activity or the like.

The substance bound to the secretory HB-EGF on the cell membrane may beany substance, so long as it is capable of binding to the secretoryHB-EGF on the cell membrane. Specifically, it includes polysaccharides,preferably glycosaminoglycan, and more preferably heparan sulfate.

HB-EGF has activity of binding to diphtheria toxin or EGF receptor ErbB1or ErbB4.

The membrane type HB-EGF includes proteins of the following (a), (b) and(c), and the like:

(a) a protein comprising the amino acid sequence represented by SEQ IDNO:2;(b) a protein consisting of an amino acid sequence in which one or moreamino acids are deleted, substituted, inserted and/or added in the aminoacid sequence represented by SEQ ID NO:2, and having activity of bindingto diphteria toxin;(c) a protein consisting of an amino acid sequence having 80% or morehomology with the amino acid sequence represented by SEQ ID NO:2, andhaving activity of binding to diphteria toxin.

Also, the secretory HB-EGF includes proteins of the following (a), (b)and (c), and the like:

(a) a protein comprising the amino acid sequence represented by SEQ IDNO:3, 4 or 5;(b) a protein consisting of an amino acid sequence in which one or moreamino acids are deleted, substituted, inserted and/or added in the aminoacid sequence in the amino acid sequence represented by SEQ ID NO:3, 4or 5, and having activity of binding to EGF receptor ErbB1 or ErbB4;(c) a protein consisting of an amino acid sequence having 80% or morehomology with the amino acid sequence represented by SEQ ID NO:3, 4 or5, and having activity of binding to EGF receptor ErbB1 or ErbB4.

In the present invention, the protein consisting of an amino acidsequence wherein one or more amino acids are deleted, substituted,inserted and/or added in the amino acid sequence represented by any oneof SEQ ID NOs:2, 3, 4 or 5 and having activity of binding to diphteriatoxin or EGF receptor ErbB1 or ErbB4, means a protein obtained forexample, by introducing a site-directed mutation into DNA encoding theprotein having the amino acid sequence represented by any one of SEQ IDNO:2, 3, 4 or 5 by site-directed mutagenesis described in MolecularCloning, Second Edition, Current Protocols in Molecular Biology(1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad.Sci., USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic AcidsResearch, 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985),or the like. The number of amino acid residues which are deleted,substituted, inserted and/or added is one or more, and is notspecifically limited, but it is within the range where deletion,substitution, insertion or addition is possible by known methods such asthe above site-directed mutagenesis. The suitable number is 1 to dozens,preferably 1 to 20, more preferably 1 to 10, and most preferably 1 to 5.

Also, the protein having 80% or more homology to the amino acid sequencerepresented by SEQ ID NO:2, 3, 4 or 5 and having activity of binding todiphteria toxin or EGR receptor ErbB1 or ErbB4 is a protein having atleast 80% or more homology, preferably 85% or more homology, morepreferably 90% or more homology, further preferably 95% or morehomology, particularly preferably 97% or more homology, and mostpreferably 99% or more homology to the amino acid sequence representedby any one of SEQ ID NO:2, 3, 4 or 5, and having activity of binding todiphteria toxin or EGR receptor ErbB1 or ErbB4.

The number of the homology described in the present invention may be aknown number calculated by using a known homology search program, unlessotherwise indicated. Regarding the nucleotide sequence, the number maybe calculated by using a default parameter in BLAST [J. Mol. Biol., 215,403 (1990)] or the like, and regarding the amino acid sequence, thenumber may be calculated by using a default parameter in BLAST2 [NucleicAcids Res., 25, 3389 (1997)], Genome Res., 7, 649 (1997) orhttp://www.ncbi.nlm.nih.gov/Education/BLASTinfo/information3.html. Asthe default parameter, G (cost to open gap) is 5 for the nucleotidesequence and 11 for the amino acid sequence; —E (cost to extend gap) is2 for the nucleotide sequence and 1 for the amino acid sequence; —q(penalty for nucleotide mismatch) is −3; —r (reward for nucleotidematch) is 1; —e (expect value) is 10; —W (wordsize) is 11 residues forthe nucleotide sequence and 3 residues for the amino acid sequence; —y(dropoff (X) for blast extensions in bits) is 20 for blastn and 25 for aprogram other than blastn(http://www.ncbi.nlm.nih.gov/blast/html/blastcgihelp.html). Also, theanalysis software for amino acid sequence includes FASTA [Methods inEnzymology, 183, 63 (1990)] and the like.

The antibody of the present invention includes a monoclonal antibodywhich binds to a cell membrane-bound HB-EGF, a membrane type HB-EGF anda secretory HB-EGF and is capable of binding to epimdermal growthfactor-like domain (EGF-like domain) of the cell membrane-bound HB-EGF,the membrane type HB-EGF and the secretory HB-EGF.

The EGF-like domain includes, for example, a polypeptide comprising theamino acid sequence represented by SEQ ID NO:4 or 5, and the like.

The monoclonal antibody which binds to the EGF-like domain includes amonoclonal antibody which inhibits binding of a secretory HB-EGF and anHB-EGF receptor.

The monoclonal antibody which inhibits binding of a secretory HB-EGF andan HB-EGF receptor includes a monoclonal antibody which binds to thebinding region of a secretory HB-EGF and an HB-EGF receptor ordiphtheria toxin, and the like.

The antibody of the present invention includes an antibody havingneutralizing activity for a secretory HB-EGF. In the present invention,the neutralizing activity is activity which inhibits biological activityof a secretory HB-EGF, and includes, for example, activity whichinhibits cell growth of a cell expressing an HB-EGF receptor, and thelike.

Examples of the antibody of the present invention include a monoclonalantibody which binds to an epitope including at least one amino acidamong amino acids at positions 115 to 147, preferably a monoclonalantibody which binds to an epitope containing at least one amino acidamong amino acids at positions 133 to 147, more preferably a monoclonalantibody which binds to an epitope containing at least one amino acidamong amino acids at positions 115, 122, 124, 125, 127, 129, 133, 135,141 and 147, still more preferably a monoclonal antibody which binds toan epitope containing at least amino acids at positions 133 and 135among amino acids at positions 133, 135 and 147, and most preferably amonoclonal antibody which binds to an epitope containing amino acids atpositions 133, 135 and 147, in the polypeptide having the amino acidsequence represented by SEQ ID NO:2, and the like.

Furthermore, examples of the antibody of the present invention include amonoclonal antibody which binds to an epitope to which a monoclonalantibody produced by hybridoma KM3566 (FERM BP-10490), a monoclonalantibody produced by hybridoma KM3567 (FERM BP-10573) or a monoclonalantibody produced by hybridoma KM3579 (FERM BP-10491) binds.

Examples of the antibody having neutralizing activity include amonoclonal antibody which binds to an epitope containing amino acids atpositions 133, 135 and 147 in the polypeptide having the amino acidsequence represented by SEQ ID NO:2.

The monoclonal antibody is an antibody secreted by a single cloneantibody-producing cell, and recognizes only one epitope (also calledantigen determinant) and has a uniform amino acid sequence (primarystructure).

The monoclonal antibody of the present invention includes an antibodyproduced by a hybridoma, a recombinant antibody and the like.

An epitope includes a single amino acid sequence, a conformationalstructure composed of amino acid sequence, a sugar chain-bound aminoacid sequence, a conformational structure composed of a sugarchain-bound amino acid sequence, etc. recognized and bound by amonoclonal antibody.

Example of the epitope include an epitope containing at least one aminoacid among amino acids at positions 115 to 147 in the amino acidsequence represented by SEQ ID NO:2, preferably an epitope containing atleast one amino acid among amino acids at positions 133 to 147, morepreferably an epitope containing at least one amino acid among aminoacids at positions 115, 122, 124, 125, 127, 129, 133, 135, 141 and 147,still more preferably an epitope containing at least amino acids atpositions 133 and 135 among amino acids at positions 133, 135 and 147,and most preferably an epitope containing amino acids at positions 133,135 and 147, in the polypeptide having the amino acid sequencerepresented by SEQ ID NO:2, and the like.

A hybridoma is a cell producing a monoclonal antibody having desiredimmuno specificity which is obtained by cell fusion of a B cell obtainedby immunizing a non-human mammal with an antigen, with a myeloma cell.

The recombinant antibody includes an antibody produced by generecombination, such as a human chimeric antibody, a humanized antibody,a human antibody and an antibody fragment thereof. Among the recombinantantibodies, one having characteristics as a monoclonal antibody, lowimmunogenecity and prolonged half-life in blood is preferable as atherapeutic agent.

Examples of the recombinant antibody of the present invention include arecombinant antibody in which CDR1, CDR2 and CDR3 of VH of the antibodycomprise the amino acid sequences represented by SEQ ID NOs:12, 13 and14, respectively, and CDR1, CDR2 and CDR3 of VL of the antibody comprisethe amino acid sequences represented by SEQ ID NOs:15, 16 and 17,respectively.

The human chimeric antibody is an antibody comprising VH and VL of anantibody of a non-human animal and a heavy chain constant region(hereinafter referred to as “CH”) and a light chain constant region(hereinafter referred to as “CL”) of a human antibody.

The human chimeric antibody of the present invention can be produced asfollows. First, cDNAs encoding VH and VL are obtained from a hybridomaproducing a monoclonal antibody which binds to a cell membrane-boundHB-EGF, a secretory HB-EGF and a membrane type HB-EGF. The resultingcDNAs are inserted into an expression vector for animal cell comprisinggenes encoding CH and CL of a human antibody to thereby construct ahuman chimeric antibody expression vector, the human chimeric antibodyexpression vector is introduced into an animal cell to thereby expressthe human chimeric antibody, and then the human chimeric antibody can beproduced.

As the CH of the human chimeric antibody, any CH can be used, so long asit belongs to a human immunoglobulin (hereinafter referred to as “hIg”),and those belonging to the hIgG class are preferred, and any one of thesubclasses belonging to the hIgG class, such as hIgG1, hIgG2, hIgG3 andhIgG4, can be used. As the CL of the human chimeric antibody, any CL canbe used, so long as it belongs to the hIg class, and those belonging tothe κ class or λ class can be used.

The human chimeric antibody of the present invention includes, forexample, a human chimeric antibody in which VH of the antibody comprisesthe amino acid sequence represented by SEQ ID NO:9 and VL of theantibody comprises the amino acid sequence represented by SEQ ID NO:11,human chimeric antibody KM3966 and the like.

A humanized antibody is an antibody in which amino acid sequences ofCDRs of VH and VL of an antibody derived from a non-human animal aregrafted into appropriate positions of VH and VL of a human antibody, andis also called a CDR-grafted antibody, a reshaped-antibody or the like.

The humanized antibody of the present invention can be produced asfollows. First, cDNAs encoding V regions in which the amino acidsequences of CDRs of VH and VL of a monoclonal antibody derived from anon-human animal which binds to a cell membrane-bound HB-EGF, asecretory HB-EGF and a membrane type HB-EGF are grafted into frameworks(hereinafter referred to as “FR”) of VH and VL of any human antibody areconstructed. The constructed cDNAs are respectively inserted into anexpression vector for animal cell comprising genes encoding CH and CL ofa human antibody to thereby construct a humanized antibody expressionvector. Next, the constructed humanized antibody expression vector isintroduced into an animal cell to thereby express the humanizedantibody, and the humanized antibody can be produced.

As the amino acid sequences of FRs of VH and VL of a human antibody, anyamino acid sequences can be used, so long as they are amino acidsequences of FRs of VH and VL, respectively, derived from a humanantibody. Examples include amino acid sequences of FRs of VH and VL ofhuman antibodies registered in database such as Protein Data Bank,common amino acid sequences of each sub group of FRs of VH and VL ofhuman antibodies described in, for example, Sequences of Proteins ofImmunological Interest, US Dept. Health and Human Services (1991), andthe like.

As the CH of the humanized antibody, any CH can be used, so long as itbelongs to the hIg, and those of the hIgG class are preferred and anyone of the subclasses belonging to the hIgG class, such as hIgG1, hIgG2,hIgG3 and hIgG4 can be used. As the CL of the human CDR-graftedantibody, any CL can be used, so long as it belongs to the hIg class,and those belonging to the κ class or λ class can be used.

The humanized antibody of the present invention includes, for example, ahumanized antibody in which VH of the antibody comprises the amino acidsequence represented by SEQ ID NO:22 or an amino acid sequence in whichat least one amino acid residue selected from Ala at position 9, Val atposition 20, Thr at position 30, Arg at position 38, Pro at position 41,Met at position 48, Arg at position 67, Val at position 68, Ile atposition 70, Tyr at position 95 and Val at position 118 in the aminoacid sequence represented by SEQ ID NO:22 is substituted with otheramino acid residue, and/or VL of the antibody comprises the amino acidsequence represented by SEQ ID NO:23 or an amino acid sequence in whichat least one amino acid residue selected from Leu at position 15, Ala atposition 19, Ile at position 21, Pro at position 49 and Leu at position84 in the amino acid sequence represented by SEQ ID NO:23 is substitutedwith other amino acid residue, and the like. The number of thesemodifications to be introduced is not particularly limited.

Examples of humanized antibodies are shown below.

For example, regarding the amino acid sequence of VH of the antibody,examples include:

a humanized antibody in which VH of the antibody comprises an amino acidsequence in which Val at position 20, Thr at position 30, Arg atposition 38, Met at position 48, Arg at position 67, Val at position 68,Ile at position 70, Tyr at position 95 and Val at position 118 in theamino acid sequence represented by SEQ ID NO:22 are substituted withother amino acid residues, preferably VH of the antibody comprises anamino acid sequence in which Val at position 20, Thr at position 30, Metat position 48, Val at position 68, Ile at position 70, Tyr at position95 and Val at position 118 are substituted with other amino acidresidues;

preferably a humanized antibody in which VH of the antibody comprises anamino acid sequence in which Thr at position 30, Met at position 48, Valat position 68, Ile at position 70 and Tyr at position 95;

preferably Thr at position 30, Met at position 48, Val at position 68and Ile at position 70 are substituted with other amino acid residues;

preferably a humanized antibody in which VH of the antibody comprises anamino acid sequence in which Thr at position 30, Val at position 68, Ileat position 70 and Tyr at position 95 are substituted with other aminoacid residues;

preferably a humanized antibody in which VH of the antibody comprises anamino acid sequence in which Thr at position 30, Val at position 68 andIle at position 70 are substituted with other amino acid residues;

preferably a humanized antibody in which VH of the antibody comprises anamino acid sequence in which Thr at position 30 and Ile at position 70are substituted with other amino acid residues; and the like.

The amino acid sequence of VH of the antibody obtained by the aboveamino acid modifications include an amino acid sequence into which atleast one modification selected from substitutions of Ala at position 9to Thr, Val at position 20 to Leu, Thr at position 30 to Arg, Arg atposition 38 to Lys, Pro at position 41 to Thr, Met at position 48 toIle, Arg at position 67 to Lys, Val at position 68 to Ala, Ile atposition 70 to Leu, Tyr at position 95 to Phe, and Val at position 118to Leu is introduced in the amino acid sequence represented by SEQ IDNO:22.

The amino acid sequence of VH into which 11 modifications are introducedincludes, for example, an amino acid sequence in which modifications arecarried out to substitute Ala at position 9 with Thr, Val at position 20with Leu, Thr at position 30 with Arg, Arg at position 38 to Lys, Pro atposition 41 with Thr, Met at position 48 with Ile, Arg at position 67with Lys, Val at position 68 with Ala, Ile at position 70 with Leu, Tyrat position 95 with Phe, and Val at position 118 with Leu in the aminoacid sequence represented by SEQ ID NO:22.

The amino acid sequence of VH into which 10 modifications are introducedincludes, for example, an amino acid sequence in which modifications arecarried out to substitute Ala at position 9 with Thr, Val at position 20with Leu, Thr at position 30 with Arg, Arg at position 38 with Lys, Proat position 41 with Thr, Met at position 48 with Ile, Arg at position 67with Lys, Val at position 68 with Ala, Ile at position 70 with Leu, andTyr at position 95 with Phe in the amino acid sequence represented bySEQ ID NO:22, and the like.

The amino acid sequence of VH into which 9 modifications are introducedincludes, for example,

an amino acid sequence in which modifications are carried out tosubstitute Ala at position 9 with Thr, Val at position 20 with Leu, Thrat position 30 with Arg, Pro at position 41 with Thr, Met at position 48with Ile, Arg at position 67 with Lys, Val at position 68 with Ala, Ileat position 70 with Leu, and Tyr at position 95 with Phe,

an amino acid sequence in which modifications are carried out tosubstitute Val at position 20 with Leu, Thr at position 30 with Arg, Argat position 38 with Lys, Met at position 48 with Ile, Arg at position 67with Lys, Val at position 68 with Ala, Ile at position 70 with Leu, Tyrat position 95 with Phe, and Val at position 118 with Leu, and the like,in the amino acid sequence of SEQ ID NO:22.

The amino acid sequence of VH into which 8 modifications are introducedincludes, for example,

an amino acid sequence in which modifications are carried out tosubstitute Ala at position 9 with Thr, Val at position 20 with Leu, Thrat position 30 with Arg, Pro at position 41 with Thr, Met at position 48with Ile, Val at position 68 with Ala, Ile at position 70 with Leu, andTyr at position 95 with Phe,

an amino acid sequence in which modifications are carried out tosubstitute Val at position 20 with Leu, Thr at position 30 with Arg, Metat position 48 with Ile, Arg at position 67 with Lys, Val at position 68with Ala, Ile at position 70 with Leu, Tyr at position 95 with Phe, andVal at position 118 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Val at position 20 with Leu, Thr at position 30 with Arg, Argat position 38 with Lys, Met at position 48 with Ile, Val at position 68with Ala, Ile at position 70 with Leu, Tyr at position 95 with Phe, andVal at position 118 with Leu, and the like, in the amino acid sequencerepresented by SEQ ID NO:22.

The amino acid sequence into which 7 modifications are introducedincludes, for example,

an amino acid sequence in which modifications are carried out tosubstitute Ala at position 9 with Thr, Thr at position 30 with Arg, Proat position 41 with Thr, Met at position 48 with Ile, Val at position 68with Ala, Ile at position 70 with Leu, and Tyr at position 95 with Phe,

an amino acid sequence in which modifications are carried out tosubstitute Val at position 20 with Leu, Thr at position 30 with Arg, Metat position 48 with Ile, Val at position 68 with Ala, Ile at position 70with Leu, Tyr at position 95 with Phe, and Val at position 118 with Leu,and the like, in the amino acid sequence represented by SEQ ID NO:22.

The amino acid sequence into which 6 modifications are introducedincludes, for example,

an amino acid sequence in which modifications are carried out tosubstitute Ala at position 9 with Thr, Thr at position 30 with Arg, Metat position 48 with Ile, Val at position 68 with Ala, Ile at position 70with Leu, and Tyr at position 95 with Phe,

an amino acid sequence in which modifications are carried out tosubstitute Val at position 20 with Leu, Thr at position 30 with Arg, Metat position 48 with Ile, Val at position 68 with Ala, Ile at position 70with Leu, and Tyr at position 95 with Phe, and the like, in the aminoacid sequence represented by SEQ ID NO:22.

The amino acid sequence of VH into which 5 modifications are introducedincludes, for example,

an amino acid sequence in which modifications are carried out tosubstitute Ala at position 9 with Thr, Thr at position 30 with Arg, Metat position 48 with Ile, Val at position 68 with Ala, and Ile atposition 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg, Met at position 48 with Ile, Valat position 68 with Ala, Ile at position 70 with Leu, and Tyr atposition 95 with Phe, and the like, in the amino acid sequence of SEQ IDNO:22.

The amino acid sequence into which 4 modifications are introducedincludes, for example,

an amino acid sequence in which modifications are carried out tosubstitute Ala at position 9 with Thr, Thr at position 30 with Arg, Valat position 68 with Ala, and Ile at position 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg, Met at position 48 with Ile, Valat position 68 with Ala, and Ile at position 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Val at position 20 with Leu, Thr at position 30 with Arg, Valat position 68 with Ala, and Ile at position 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg, Arg at position 38 with Lys, Valat position 68 with Ala, and Ile at position 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg, Pro at position 41 with Thr, Valat position 68 with Ala, and Ile at position 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg, Arg at position 67 with Lys, Valat position 68 with Ala, and Ile at position 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg, Val at position 68 with Ala, Ileat position 70 with Leu, and Tyr at position 95 with Phe,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg, Val at position 68 with Ala, Heat position 70 with Leu, and Val at position 118 with Leu, and the like,in the amino acid sequence represented by SEQ ID NO:22.

The amino acid sequence of VH into which three modifications areintroduced includes, for example,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg, Val at position 68 with Ala, andIle at position 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Ala at position 9 with Thr, Thr at position 30 with Arg, andIle at position 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Val at position 20 with Leu, Thr at position 30 with Arg, andIle at position 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg, Arg at position 38 with Lys, andIle at position 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg, Pro at position 41 with Thr, andIle at position 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg, Met at position 48 with Ile, andIle at position 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg, Arg at position 67 with Lys, andIle at position 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg, Ile at position 70 with Leu, andTyr at position 95 with Phe,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg, Ile at position 70 with Leu, andVal at position 118 with Leu, and the like, in the amino acid sequencerepresented by SEQ ID NO:22.

The amino acid sequence of VET into which two modifications areintroduced includes, for example,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg and Ile at position 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Ala at position 9 with Thr and Ile at position 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Val at position 20 with Leu and Ile at position 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Arg at position 38 with Lys and Ile at position 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Pro at position 41 with Thr and Ile at position 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Met at position 48 with Ile and Ile at position 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Arg at position 67 with Lys and Ile at position 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Val at position 68 with Ala and Ile at position 70 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Ile at position 70 with Leu and Tyr at position 95 with Phe,

an amino acid sequence in which modifications are carried out tosubstitute Ile at position 70 with Leu and Val at position 118 with Leu,

an amino acid sequence in which modifications are carried out tosubstitute Ala at position 9 with Thr and Thr at position 30 with Arg,

an amino acid sequence in which modifications are carried out tosubstitute Val at position 20 with Leu and Thr at position 30 with Arg,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg and Arg at position 38 with Lys,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg and Pro at position 41 with Thr,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg and Met at position 48 with Ile,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg and Arg at position 67 with Lys,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg and Val at position 68 with Ala,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg and Tyr at position 95 with Phe,

an amino acid sequence in which modifications are carried out tosubstitute Thr at position 30 with Arg and Val at position 118 with Leu,and the like, in the amino acid sequence represented by SEQ ID NO:22.

The amino acid sequence of VH into which one modification is introducedincludes, for example,

an amino acid sequence in which Ala at position 9 is substituted withThr,

an amino acid sequence in which Val at position 20 is substituted withLeu,

an amino acid sequence in which Thr at position 30 is substituted withArg,

an amino acid sequence in which Arg at position 38 is substituted withLys,

an amino acid sequence in which Pro at position 41 is substituted withThr,

an amino acid sequence in which Met at position 48 is substituted withIle,

an amino acid sequence in which Arg at position 67 is substituted withLys,

an amino acid sequence in which Val at position 68 is substituted withAla,

an amino acid sequence in which Ile at position 70 is substituted withIle,

an amino acid sequence in which Tyr at position 95 is substituted withPhe, and

an amino acid sequence in which Val at position 118 is substituted withLeu

in the amino acid sequence of SEQ ID NO:22.

VL of the antibody includes, for example, an amino acid sequence inwhich Leu at position 15, Ala at position 19, Ile at position 21 and Leuat position 84 are substituted in the amino acid sequence represented bySEQ ID NO:23.

It is preferably an amino acid in which Ala at position 19, Ile atposition 21 and Leu at position 84 are substituted.

The amino acid sequence obtained by the above amino acid modificationsinclude an amino acid sequence into which at least one modificationselected from substitutions of Leu at position 15 with Val, Ala atposition 19 with Val, Ile at position 21 with Met, Pro at position 49with Ser, and Leu at position 84 with Val in the amino acid sequencerepresent by SEQ ID NO:23 is introduced.

The amino acid sequence of VL into which 5 modifications are introducedincludes, for example, an amino acid sequence in which modifications arecarried out to substitute Leu at position 15 with Val, Ala at position19 with Val, Ile at position 21 with Met, Pro at position 49 with Ser,and Leu at position 84 with Val, and the like, in the amino acidsequence represented by SEQ ID NO:23.

The amino acid sequence of VL into which 4 modifications are introducedincludes, for example,

an amino acid sequence in which modifications are carried out tosubstitute Leu at position 15 with Val, Ala at position 19 with Val, Ileat position 21 with Met, and Pro at position 49 with Ser,

an amino acid sequence in which modifications are carried out tosubstitute Leu at position 15 with Val, Ala at position 19 with Val, Ileat position 21 with Met, and Leu at position 84 with Val,

an amino acid sequence in which modifications are carried out tosubstitute Leu at position 15 with Val, Ala at position 19 with Val, Proat position 49 with Ser, and Leu at position 84 with Val,

an amino acid sequence in which modifications are carried out tosubstitute Leu at position 15 with Val, Ile at position 21 with Met, Proat position 49 with Ser, and Leu at position 84 with Val,

an amino acid sequence in which modifications are carried out tosubstitute Ala at position 19 with Val, Ile at position 21 with Met, Proat position 49 with Ser, and Leu at position 84 with Val, and the like,

in the amino acid sequence represented by SEQ ID NO:23.

The amino acid sequence of VL into which three modifications areintroduced includes, for example,

an amino acid sequence in which modifications are carried out tosubstitute Leu at position 15 with Val, Ala at position 19 with Val, andIle at position 21 with Met,

an amino acid sequence in which modifications are carried out tosubstitute Leu at position 15 with Val, Ala at position 19 with Val, andPro at position 49 with Ser,

an amino acid sequence in which modifications are carried out tosubstitute Leu at position 15 with Val, Ala at position 19 with Val, andLeu at position 84 with Val,

an amino acid sequence in which modifications are carried out tosubstitute Leu at position 15 with Val, Ile at position 21 with Met, andPro at position 49 with Ser,

an amino acid sequence in which modifications are carried out tosubstitute Leu at position 15 with Val, Ile at position 21 with Met, andLeu at position 84 with Val,

an amino acid sequence in which modifications are carried out tosubstitute Leu at position 15 with Val, Pro at position 49 with Ser, andLeu at position 84 with Val,

an amino acid sequence in which modifications are carried out tosubstitute Ala at position 19 with Val, Ile at position 21 with Met, andPro at position 49 with Ser,

an amino acid sequence in which modifications are carried out tosubstitute Ala at position 19 with Val, Ile at position 21 with Met, andLeu at position 84 with Val,

an amino acid sequence in which modifications are carried out tosubstitute Ala at position 19 with Val, Pro at position 49 with Ser, andLeu at position 84 with Val,

an amino acid sequence in which modifications are carried out tosubstitute Ile at position 21 with Met, Pro at position 49 with Ser, andLeu at position 84 with Val, and the like, in the amino acid sequencerepresented by SEQ ID NO:23.

The amino acid sequence of VL into which two modifications areintroduced includes, for example,

an amino acid sequence in which modifications are carried out tosubstitute Leu at position 15 with Val and Ala at position 19 with Val,

an amino acid sequence in which modifications are carried out tosubstitute Leu at position 15 with Val and Ile at position 21 with Met,

an amino acid sequence in which modifications are carried out tosubstitute Leu at position 15 with Val and Pro at position 49 with Ser,

an amino acid sequence in which modifications are carried out tosubstitute Leu at position 15 with Val and Leu at position 84 with Val,

an amino acid sequence in which modifications are carried out tosubstitute Ala at position 19 with Val and Ile at position 21 with Met,

an amino acid sequence in which modifications are carried out tosubstitute Ala at position 19 with Val and Pro at position 49 with Ser,

an amino acid sequence in which modifications are carried out tosubstitute Ala at position 19 with Val and Leu at position 84 with Val,

an amino acid sequence in which modifications are carried out tosubstitute Ile at position 21 with Met and Pro at position 49 with Ser,

an amino acid sequence in which modifications are carried out tosubstitute Ile at position 21 with Met and Leu at position 84 with Val,

an amino acid sequence in which modifications are carried out tosubstitute Pro at position 49 with Ser and Leu at position 84 with Val,and the like, in the amino acid sequence represented by SEQ ID NO:23.

The amino acid sequence of VL into which one modification is introducedincludes, for example,

an amino acid sequence in which Leu at position 15 is substituted withVal,

an amino acid sequence in which Ala at position 19 is substituted withVal,

an amino acid sequence in which Ile at position 21 is substituted withMet,

an amino acid sequence in which Pro at position 49 is substituted withSer,

an amino acid sequence in which Leu at position 84 is substituted withVal, and the like, in the amino acid sequence represented by SEQ IDNO:23.

Specific examples of the humanized antibody of the present inventioninclude a humanized antibody wherein H chain of variable regioncomprises the amino acid sequence represented by SEQ ID NO:22 and/or Lchain of variable region comprises the amino acid sequence representedby SEQ ID NO:23; a humanized antibody wherein H chain of variable regioncomprises the amino acid sequence represented by SEQ ID NO:22 and/or Lchain of variable region comprises the amino acid sequence representedby SEQ ID NO:43; a humanized antibody wherein H chain of variable regioncomprises the amino acid sequence represented by SEQ ID NO:42 and/or Lchain of variable region comprises the amino acid sequence representedby SEQ ID NO:23; a humanized antibody wherein H chain of variable regioncomprises the amino acid sequence represented by SEQ ID NO:42 and/or Lchain of variable region comprises the amino acid sequence representedby SEQ ID NO:43; and the like.

A human antibody is originally an antibody naturally existing in thehuman body, but it also includes antibodies obtained from a humanantibody phage library or a human antibody-producing transgenic animal,which is prepared based on the recent advance in genetic engineering,cell engineering and developmental engineering techniques. The antibodyexisting in the human body can be prepared, for example by isolating ahuman peripheral blood lymphocyte, immortalizing it by infecting with EBvirus or the like and then cloning it to thereby obtain lymphocytescapable of producing the antibody, culturing the lymphocytes thusobtained, and purifying the antibody from the supernatant of theculture. The human antibody phage library is a library in which antibodyfragments such as Fab and scFv are expressed on the phage surface byinserting a gene encoding an antibody prepared from a human B cell intoa phage gene. A phage expressing an antibody fragment having the desiredantigen binding activity can be recovered from the library, using itsactivity to bind to an antigen-immobilized substrate as the index. Theantibody fragment can be converted further into a human antibodymolecule comprising two full H chains and two full L chains by geneticengineering techniques. A human antibody-producing transgenic animal isan animal in which a human antibody gene is integrated into cells.Specifically, a human antibody-producing transgenic animal can beprepared by introducing a gene encoding a human antibody into a mouse EScell, grafting the ES cell into an early stage embryo of other mouse andthen developing it. A human antibody is prepared from the humanantibody-producing transgenic non-human animal by obtaining a humanantibody-producing hybridoma by a hybridoma preparation method usuallycarried out in non-human mammals, culturing the obtained hybridoma andforming and accumulating the human antibody in the supernatant of theculture.

In the amino acid sequence constituting the above antibody or antibodyfragment, an antibody or antibody fragment thereof in which one or moreamino acids are deleted, substituted, inserted or added, having activitysimilar to the above antibody or the antibody fragment thereof is alsoincluded in the antibody or the antibody fragment thereof of the presentinvention.

The number of amino acids which are deleted, substituted, insertedand/or added is one or more, and is not specifically limited, but it iswithin the range where deletion, substitution or addition is possible byknown methods such as the site-directed mutagenesis described inMolecular Cloning, Second Edition, Current Protocols in MolecularBiology, Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci.USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13,4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985), or the like.For example, the number is 1 to dozens, preferably 1 to 20, morepreferably 1 to 10, and most preferably 1 to 5.

The expression “one or more amino acids are deleted, substituted,inserted or added” in the amino acid sequence of the above antibodymeans the followings. That is, it means there is deletion, substitution,insertion or addition of one or plural amino acids at optional positionsin the same sequence and one or plural amino acid sequences. Also, thedeletion, substitution, insertion or addition may occur at the same timeand the amino acid which is substituted, inserted or added may be eithera natural type or a non-natural type. The natural type amino acidincludes L-alanine, L-asparagine, L-aspartic acid, L-glutamine,L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine,L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine,L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and thelike.

Preferable examples of mutually substitutable amino acids are shownbelow. The amino acids in the same group are mutually substitutable.

-   Group A: leucine, isoleucine, norleucine, valine, norvaline,    alanine, 2-aminobutanoic acid, methionine, O-methylserine,    t-butylglycine, t-butylalanine, cyclohexylalanine-   Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic    acid, 2-aminoadipic acid, 2-aminosuberic acid-   Group C: asparagine, glutamine-   Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid,    2,3-diaminopropionic acid-   Group E: proline, 3-hydroxyproline, 4-hydroxyproline-   Group F: serine, threonine, homoserine-   Group G: phenylalanine, tyrosine

The antibody fragment of the present invention includes Fab, Fab′,F(ab′)₂, scFv, diabody, dsFv, peptide comprising six CDRs and the like.

An Fab is obtained by treating an IgG antibody molecule with a protease,papain. This is an antibody fragment having a molecular weight of about50,000 and having antigen binding activity, in which about a half of theN-terminal side of H chain and the entire L chain, among fragmentsobtained, papain (cleaved at an amino acid residue at position 224 ofthe H chain), are bound together through a disulfide bond.

The Fab of the present invention can be produced by treating an antibodywith a protease, papain. Also, the Fab of the present invention can beproduced by inserting DNA encoding Fab of the antibody into anexpression vector for prokaryote or an expression vector for eukaryote,and introducing the vector into a prokaryote or eukaryote to express theFab.

A F(ab′)₂ is an antibody fragment having antigen binding activity andhaving a molecular weight of about 100,000 which is somewhat larger thanone in which Fab is bound via a disulfide bond in the hinge region,among fragments obtained by treating an IgG antibody molecule with aprotease, pepsin (cleaved at an amino acid residue at position 234 ofthe H chain).

The F(ab′)₂ of the present invention can be produced by treating anantibody with a protease, pepsin. Also, the F(ab′)₂ of the presentinvention can be produced by binding Fab′ described below via athioether bond or a disulfide bond.

A Fab′ is an antibody fragment having antibody binding activity andhaving a molecular weight of about 50,000 in which the disulfide bond inthe hinge region of the above F(ab′)₂ is cleaved.

The Fab′ of the present invention can be produced by F(ab′)₂ with areducing agent, dithiothreitol. Also, the Fab′ of the present inventioncan be produced by inserting DNA encoding Fab′ fragment of the antibodyinto an expression vector for prokaryote or an expression vector foreukaryote, and introducing the vector into a prokaryote or eukaryote toexpress the Fab′.

An scFv is an antibody fragment having antigen binding activity which isa VH-P-VL or VL-P-VH polypeptide in which one chain VH and one chain VLare linked using an appropriate peptide linker (hereinafter referred toas “P”). The scFv of the present invention can be produced by obtainingcDNAs encoding VH and VL of the antibody, constructing DNA encodingscFv, inserting DNA encoding scFv of the antibody into an expressionvector for prokaryote or an expression vector for eukaryote, and thenintroducing the expression vector into a prokaryote or eukaryote toexpress the scFv.

A diabody is an antibody fragment having divalent antigen bindingactivity in which scFvs are dimerized. The divalent antigen bindingactivity may be the same or different with each other. The diabody ofthe present invention can be produced by obtaining cDNAs encoding VH andVL of the antibody, constructing DNA encoding scFv so that the length ofthe amino acid sequence of P is 8 or less residues, inserting the DNAinto an expression vector for prokaryote or an expression vector foreukaryote, and then introducing the expression vector into a prokaryoteor eukaryote to express the diabody.

A dsFv is obtained by binding polypeptides in which one amino acidresidue of each of VH and VL is substituted with a cysteine residue viaa disulfide bond between the cysteine residues. The amino acid residueto be substituted with a cysteine residue can be selected based on athree-dimensional structure estimation of the antibody in accordancewith the method shown by Reiter et al. (Protein Engineering, 7, 697-704(1994)). The dsFv of the present invention can be produced by obtainingcDNAs encoding VH and VL of the antibody, constructing DNA encodingdsFv, inserting the DNA into an expression vector for prokaryote or anexpression vector for eukaryote, and then introducing the expressionvector into a prokaryote or eukaryote to express the dsFv.

The peptide comprising CDRs used in the present invention can beproduced by constructing cDNAs encoding CDRs of VH and VL of theantibody which specifically binds to HB-EGF, inserting the cDNAs into anexpression vector for prokaryote or an expression vector for eukaryote,and then introducing the expression vector into a prokaryote oreukaryote to express the peptide. The peptide comprising CDRs can alsobe produced by a chemical synthesis method such as an Fmoc method(fluorenylmethoxycarbonyl method), a tBoc method (t-butyloxycarbonylmethod), or the like.

Antibody derivatives in which a radioisotope, a protein or an agent isconjugated to the above-described antibody or the antibody fragmentthereof can be used in the present invention.

The antibody derivatives of the present invention can be produced bychemically conjugating an agent to the N-terminal side or C-terminalside of an H chain or an L chain of the antibody or the antibodyfragment thereof which binds to a cell membrane-bound HB-EGF, a membranetype HB-EGF and a secretory HB-EGF, an appropriate substituent or sidechain of the antibody or a sugar chain in the antibody [AntibodyEngineering Handbook, edited by Osamu Kanemitsu, published by ChijinShokan (1994)].

Also, the antibody derivatives can be genetically produced by linking aDNA encoding the antibody or the antibody fragment thereof which bindsto a cell membrane-bound HB-EGF, a membrane type HB-EGF and a secretoryHB-EGF to other DNAs encoding an agent, such as a protein, to be bound,inserting the DNA into a vector for expression, and introducing theexpression vector into a host cell.

The agent includes a chemotherapeutic agent, a therapeutic antibody, animmunostimulator, an agent having a high molecular weight and the like.

The protein includes cytokine, a growth factor, a toxic protein, and thelike.

Furthermore, the agent to be bound to the antibody or the antibodyfragment thereof may be in a form of a prodrug. The prodrug in thepresent invention is an agent which is subjected to chemicalmodification by an enzyme existing in the tumor environment and isconverted to a substance having an activity of damaging the tumor cells.

The chemotherapeutic agent includes any chemotherapeutic agents such asan alkylating agent, a nitrosourea agent, a metabolism antagonist, ananticancer antibiotic substance, an alkaloid derived from a plant, atopoisomerase inhibitor, an agent for hormonotherapy, a hormoneantagonist, an aromatase inhibitor, a P glycoprotein inhibitor, aplatinum complex derivative, an M-phase inhibitor and a kinaseinhibitor. Examples of the chemotherapeutic agent include amifostine(Ethyol), cisplatin, dacarbazine (DTIC), dactinomycin, mecloretamin(nitrogen mustard), streptozocin, cyclophosphamide, iphosphamide,carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin),doxorubicin lipo (Doxyl), epirubicin, gemcitabine (Gemsal),daunorubicin, daunorubicin lipo (Daunozome), procarbazine, mitomycin,cytarabine, etoposide, methotrexate, 5-fluorouracil, fluorouracil,vinblastine, vincristine, bleomycin, daunomycin, peplomycin,estramustine, paclitaxel (Taxol), docetaxel (Taxotea), aldesleukin,asparaginase, busulfan, carboplatin, oxaliplatin, nedaplatin,cladribine, camptothecin, CPT-11, 10-hydroxy-7-ethylcamptothecin (SN38),floxuridine, fludarabine, hydroxyurea, iphosphamide, idarubicin, mesna,irinotecan, nogitecan, mitoxantrone, topotecan, leuprolide, megestrol,melfalan, mercaptopurine, hydroxycarbamide, plicamycin, mitotane,pegasparagase, pentostatin, pipobroman, streptozocin, tamoxifen,goserelin, leuprorelin, flutamide, teniposide, testolactone,thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil,hydrocortisone, prednisolone, methylprednisolone, vindesine, nimustine,semustine, capecitabine, Tomudex, azacytidine, UFT, oxaliplatin,gefitinib (Iressa), imatinib (STI 571), elrotinib, Flt3 inhibitor,vascular endothelial growth factor receptor (VEGFR) inhibitor,fibroblast growth factor receptor (FGFR) inhibitor, EGFR inhibitor(Iressa, Tarceva), radicicol, 17-allylamino-17-demethoxygeldanamycin,rapamycin, amsacrine, all-trans-retinoic acid, thalidomide, anastrozole,fadrozole, letrozole, exemestane, gold thiomalate, D-penicillamine,bucillamine, azathioprine, mizoribine, cyclosporine, rapamycin,hydrocortisone, bexarotene (Targretin), tamoxifen, dexamethasone,progestin substances, estrogen substances, anastrozole (Arimidex),Leuplin, aspirin, indomethacin, celecoxib, azathioprine, penicillamine,gold thiomalate, chlorpheniramine maleate, chlorpheniramine, clemastine,tretinoin, bexarotene, arsenic, voltezomib, allopurinol, gemtuzumab,ibritumomab tiuxetan, 131 tositumomab, Targretin, ONTAK, ozogamine,clarithromycin, leucovorin, ifosfamide, ketoconazole, aminoglutethimide,suramin, methotrexate, maytansinoid and derivatives thereof, and thelike.

The method for conjugating the chemotherapeutic agent with the antibodyinclude a method in which the chemotherapeutic agent and an amino groupof the antibody are conjugated via glutaraldehyde, a method in which anamino group of the chemotherapeutic agent and a carboxyl group of theantibody are bound via a water-soluble carbodiimide, and the like.

The therapeutic antibody includes an antibody against an antigen inwhich apoptosis is induced by binding of the antibody, an antibodyagainst an antigen participating in formation of morbid state of tumorsuch as growth or metastasis of tumor cells, an antibody which regulatesimmunological function and an antibody which inhibits angiogenesis inthe morbid part.

The antigen in which apoptosis is induced by binding of the antibodyincludes cluster of differentiation (hereinafter “CD”) 19, CD20, CD21,CD22, CD23, CD24, CD37, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77,CDw78, CD79a, CD79b, CD80 (B7.1), CD81, CD82, CD83, CDw84, CD85, CD86(B7.2), human leukocyte antigen (HLA)-Class II, EGFR and the like.

The antigen for the antibody which relates to the formation ofpathological condition of tumor or regulates immunological functionincludes CD4, CD40, CD40 ligand, B7 family molecule (CD80, CD86, CD274,B7-DC, B7-H2, B7-H3, B7-H4), ligand of B7 family molecule (CD28, CTLA-4,ICOS, PD-1, BTLA), OX-40, OX-40 ligand, CD137, tumor necrosis factor(TNF) receptor family molecule (DR4, DR5, TNFR1, TNFR2), TNF-relatedapoptosis-inducing ligand receptor (TRAIL) family molecule, receptorfamily of TRAIL family molecule (TRAIL-R1, TRAIL-R2, TRAIL-R3,TRAIL-R4), receptor activator of nuclear factor kappa B ligand (RANK),RANK ligand, CD25, folic acid receptor 4, cytokine [interleukin-1α(hereinafter interleukin is referred to as “IL”), IL-1β, IL-4, IL-5,IL-6, IL-10, IL-13, transforming growth factor (TGF) β, TNFα, etc.],receptors of these cytokines, chemokine (SLC, ELC, I-309, TARC, MDC,CTACK, etc.) and receptors of these chemokines.

The antigen for the antibody which inhibits angiogenesis in the morbidpart includes vascular endothelial growth factor (VEGF), fibroblastgrowth factor (FGF), EGF, platelet-derived growth factor (PDGF),insulin-like growth factor (IGF), erythropoietin (EPO), TGFβ, IL-8,ephilin, SDF-1 and the like.

The immuno stimulator may be any natural products known asimmunoadjuvants. Examples of an agent enhancing immunogen includeβ(1→3)glucan (lentinan, schizophyllan), α-galactosylceramide (KRN7000),fungus powder (picibanil, BCG) and fungus extract (krestin).

The agent having high molecular weight includes polyethylene glycol(hereinafter referred to as “PEG”), albumin, dextran, polyoxyethylene,styrene-maleic acid copolymer, polyvinylpyrrolidone, pyran copolymer,hydroxypropylmethacrylamide, and the like. By binding these compoundshaving high molecular weight to an antibody or antibody fragment, thefollowing effects are expected: (1) improvement of stability againstvarious chemical, physical or biological factors, (2) remarkableprolongation of half-life in blood, (3) disappearance of immunogenicity,suppression of antibody production, and the like [Bioconjugate Drug,Hirokawa Shoten (1993)]. For example, the method for binding PEG to anantibody includes a method in which an antibody is allowed to react witha PEG-modifying reagent [Bioconjugate Drug, Hirokawa Shoten (1993)]. ThePEG-modifying reagent includes a modifying agent of ε-amino group oflysine (Japanese Published Unexamined Patent Application No. 178926/86),a modifying agent of a carboxyl group of aspartic acid and glutamic acid(Japanese Published Unexamined Patent Application No. 23587/81), amodifying agent of a guanidino group of arginine (Japanese PublishedUnexamined Patent Application No. 117920/90) and the like.

The cytokine or the growth factor may be any cytokine or growth factor,so long as it enhances cells such as NK cells, macrophages andneutrophils. Examples include interferon (hereinafter referred to as“INF”)-α, INF-β, INF-γ, IL-2, IL-12, IL-15, IL-18, IL-21, IL-23,granulocyte-colony stimulating factor (G-CSF), granulocytemacrophage-colony stimulating factor (GM-CSF), macrophage-colonystimulating factor (M-CSF) and the like.

The toxic protein includes ricin, diphtheria toxin, ONTAK and the like,and also includes a toxic protein wherein mutation is introduced into aprotein in order to control the toxicity.

The radioisotope includes ¹³¹I, ¹²⁵I, ⁹⁰Y, ⁶⁴Cu, ⁹⁹Tc, ⁷⁷Lu, ²¹¹At andthe like. The radioisotope can directly be conjugated with the antibodyby Chloramine-T method. Also, a substance chelating the radioisotope canbe conjugated with the antibody. The chelating agent includesmethylbenzyldiethylene-triaminepentaacetic acid (MX-DTPA) and the like.

In the present invention, the antibody used in the present invention canbe administered in combination with one or more of other agents, andradiation irradiation can be also used in combination. The other agentincludes the above-described chemotherapeutic agent, therapeuticantibody, immunostimulator, cytokine, growth factor and the like.

The radiation irradiation include photon (electromagnetic) irradiationsuch as X-ray or γ-ray, particle irradiation such as electron beam,proton beam or heavy particle bema, and the like.

In the method for combined administration, the agent may besimultaneously administered with the antibody used in the presentinvention, or the agent may be administered before or after theadministration of the antibody used in the present invention.

The present invention is described below in detail.

1. Process for Producing Recombinant Antibody Composition (1)Preparation of Antigen

An expression vector comprising cDNA encoding the secretory HB-EGF or apartial length of the secretory HB-EGF (hereinafter simply referred toas the secretory HB-EGF) is introduced into Escherichia coli, yeast, aninsect cell, an animal cell or the like for expression to obtain thesecretory HB-EGF or a partial fragment of the secretory HB-EGF. Also,HB-EGF in the extracellular region can be purified from cells expressingHB-EGF by protease treatment. The secretory HB-EGF can be purified fromvarious human tumor culturing cells, human tissue and the like whichexpress a large amount of the secretory HB-EGF. Furthermore, a syntheticpeptide having a partial sequence of the secretory HB-EGF can beprepared and used as an antigen.

Specifically, the secretory HB-EGF used in the present invention can beproduced, for example, by expressing a DNA encoding it in a host cellusing a method described in Molecular Cloning, A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press (1989), CurrentProtocols in Molecular Biology, John Wiley & Sons (1987-1997) or thelike as follows.

Firstly, a recombinant vector is produced by inserting a full lengthcDNA into downstream of a promoter of an appropriate expression vector.At this time, if necessary, a DNA fragment having an appropriate lengthcontaining a region encoding the polypeptide based on the full lengthcDNA may be prepared, and the DNA fragment may be used instead of theabove full length cDNA. Next, a transformant producing HB-EGF can beobtained by introducing the recombinant vector into a host cell suitablefor the expression vector.

The host cell can be any cell so long as it can express the gene ofinterest, and includes Escherichia coli, an animal cell and the like.

The expression vector includes vectors which can replicate autonomouslyin the host cell to be used or vectors which can be integrated into achromosome comprising an appropriate promoter at such a position thatthe DNA encoding the secretory HB-EGF can be transcribed.

When a prokaryote such as Escherichia coli is used as the host cell, itis preferred that the recombinant vector comprising the DNA encodingHB-EGF used in the present invention is autonomously replicable in theprokaryote and contains a promoter, a ribosome binding sequence, the DNAused in the present invention and a transcription termination sequence.The recombinant vector may further comprise a gene regulating thepromoter.

The expression vector includes, for example, pBTrp2, pBTac1, pBTac2 (allmanufactured by Roche Diagnostics), pKK233-2 (manufactured byPharmacia), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufacturedby Promega), pQE-8 (manufactured by QIAGEN), pKYP10 (Japanese PublishedUnexamined Patent Application No. 110600/83), pKYP200 [AgriculturalBiological Chemistry, 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53,277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)],pBluescript II SK(−) (manufactured by Stratagene), pTrs30 [prepared fromEscherichia coli JM109/pTrS30 (FERM BP-5407)], pTrs32 [prepared fromEscherichia coli JM109/pTrS32 (FERM BP-5408)], pGHA2 [prepared fromEscherichia coli IGHA2 (FERM BP-400), Japanese Published UnexaminedPatent Application No. 221091/85], pGKA2 [prepared from Escherichia coliIGKA2 (FERM BP-6798), Japanese Published Unexamined Patent ApplicationNo. 221091/85], pTerm2 (U.S. Pat. No. 4,686,191, U.S. Pat. No.4,939,094, U.S. Pat. No. 5,160,735), pSupex, pUB110, pTP5, pC194, pEG400[J. Bacteriol., 172, 2392 (1990)], pGEX (manufactured by Pharmacia), pETsystem (manufactured by Novagen), pME18SFL3 and the like.

Any promoter can be used, so long as it can function in the host cell tobe used. Examples include promoters derived from Escherichia coli, phageand the like, such as trp promoter (Ptrp), lac promoter, PL promoter, PRpromoter and T7 promoter. Also, artificially designed and modifiedpromoters, such as a tandem promoter in which two Ptrp are linked intandem, tac promoter, lacT7 promoter and letI promoter, can be used.

Also, the above recombinant vector is preferably a plasmid in which thespace between Shine-Dalgarno sequence, which is the ribosome bindingsequence, and the initiation codon is adjusted to an appropriatedistance (for example, 6 to 18 nucleotides). In the nucleotide sequenceof DNA encoding the secretory HB-EGF used in the present invention,nucleotides can be arranged so as to obtain a suitable codon forexpression in the host so that the producing ratio of the secretoryHB-EGF of interest can be improved. Furthermore, the transcriptiontermination sequence is not essential to express a gene in the aboverecombinant vector. However, it is preferred to arrange a transcriptionterminating sequence immediately downstream of the structural gene.

The prokaryotes used for the host cells include prokaryotes belonging tothe genera Escherichia, and examples include Escherichia coli XL1-Blue,Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli DH5α,Escherichia coli BL21 (DE3), Escherichia coliMC1000, Escherichia coliKY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coliHB101, Escherichia coli No. 49, Escherichia coli W3110, Escherichia coliNY49 and the like.

Any introduction method of the recombinant vector can be used, so longas it is a method for introducing DNA into the above-described hostcell, and examples include a method using a calcium ion described inProc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982),Molecular & General Genetics, 168, 111 (1979) and the like. When ananimal cell is used as the host cell, an expression vector includes, forexample, pcDNAI, pcDM8 (available from Funakoshi), pAGE107 [JapanesePublished Unexamined Patent Application No. 22979/91; Cytotechnology, 3,133 (1990)], pAS3-3 (Japanese Published Unexamined Patent ApplicationNo. 227075/90), pCDM8 [Nature, 329, 840, (1987)], pcDNAI/Amp(manufactured by Invitrogen), pREP4 (manufactured by Invitrogen),pAGE103 [J. Biochemistry, 101, 1307 (1987)], pAGE210, pME18SFL3 and thelike.

Any promoter can be used, so long as it can function in an animal cell.Examples include a promoter of IE (immediate early) gene ofcytomegalovirus (CMV), SV40 early promoter, a promoter of retrovirus, ametallothionein promoter, a heat shock promoter, SRα promoter and thelike. Also, the enhancer of the IE gene of human CMV can be usedtogether with the promoter.

The host cell includes human Namalwa cell, monkey COS cell, Chinesehamster ovary (CHO) cell, HST5637 (Japanese Published Unexamined PatentApplication No. 299/88) and the like.

Any introduction method of the recombinant vector can be used, so longas it is a method for introducing DNA into an animal cell, and examplesinclude electroporation [Cytotechnology, 3, 133 (1990)], the calciumphosphate method (Japanese Published Unexamined Patent Application No.227075/90), the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413(1987)], and the like.

As the expression method of the gene, in addition to direct expression,secretory production, fusion protein expression and the like inaccordance with the method described in Molecular Cloning, A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press (1989) canbe carried out. When expression is carried out in a cell derived fromeukaryote, the secretory HB-EGF to which a sugar or a sugar chain isadded can be obtained.

The secretory HB-EGF used in the present invention can be produced byculturing the thus obtained transformant in a medium to form andaccumulate the secretory HB-EGF in the culture, and recovering it fromthe culture. The method for culturing the transformant in the medium iscarried out according to the usual method used in culturing of hosts.

When a microorganism transformed with a recombinant vector containing aninducible promoter as a promoter is cultured, an inducer can be added tothe medium, if necessary. For example,isopropyl-β-D-thiogalactopyranoside or the like can be added to themedium when a microorganism transformed with a recombinant vector usinglac promoter is cultured, or indoleacrylic acid or the like can be addedthereto when a microorganism transformed with a recombinant vector usingtrp promoter is cultured.

When a transformant obtained using an animal cell as the host cell iscultured, the medium includes generally used RPMI 1640 medium [TheJournal of the American Medical Association, 199, 519 (1967)], Eagle'sMEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEM medium[Virology, 8, 396 (1959)] and 199 medium [Proceeding of the Society forExperimental Biology and Medicine, 73, 1 (1950)], the media to whichfetal calf serum, etc. is added, and the like. The culturing is carriedout generally at a pH of 6 to 8 and 30 to 40° C. for 1 to 7 days in thepresence of 5% CO₂. If necessary, an antibiotic such as kanamycin orpenicillin can be added to the medium during the culturing.

Thus, the secretory HB-EGF used in the present invention can be producedby culturing a transformant derived from a microorganism, an animal cellor the like which comprises a recombinant vector into which a DNAencoding the secretory HB-EGF used in the present invention is inserted,in accordance with a general culturing method, to thereby form andaccumulate the polypeptide, and then recovering the secretory HB-EGFfrom the culture.

Regarding the expression method of gene, in addition to directexpression, secretory production, fusion protein expression and the likecan be carried out according to the method described in MolecularCloning, A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press (1989).

The process for producing the secretory HB-EGF includes a method ofintracellular expression in a host cell, a method of extracellularsecretion from a host cell, a method of producing on a host cellmembrane outer envelope, and the like. The appropriate method can beselected by changing the host cell used and the structure of thesecretory HB-EGF produced.

When the secretory HB-EGF is produced in a host cell or on a host cellmembrane outer envelope, the gene product can be positively secretedextracellularly in accordance with the method of Paulson et al. [J.Biol. Chem., 264, 17619 (1989)], the method of Lowe et al. [Proc. Natl.Acad. Sci. USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], themethods described in Japanese Published Unexamined Patent ApplicationNo. 336963/93 and WO94/23021, and the like.

Also, the production amount can be increased in accordance with themethod described in Japanese Published Unexamined Patent Application No.227075/90 utilizing a gene amplification system using a dihydrofolatereductase gene. The secretory HB-EGF can be isolated and purified fromthe above culture, for example, as follows. When the secretory HB-EGF isintracellularly expressed in a dissolved state, the cells afterculturing are recovered by centrifugation, suspended in an aqueousbuffer and then disrupted using ultrasonicator, French press, MantonGaulin homogenizer, dynomill or the like to obtain a cell-free extract.The cell-free extract is centrifuged to obtain a supernatant, and apurified preparation can be obtained by subjecting the supernatant to ageneral enzyme isolation and purification techniques such as solventextraction; salting out with ammonium sulfate etc.; desalting;precipitation with an organic solvent; anion exchange chromatographyusing a resin such as diethylaminoethyl (DEAE)-sepharose, DIAION HPA-75(manufactured by Mitsubishi Chemical); cation exchange chromatographyusing a resin such as S-Sepharose FF (manufactured by Pharmacia);hydrophobic chromatography using a resin such as butyl-Sepharose orphenyl-Sepharose; gel filtration using a molecular sieve; affinitychromatography; chromatofocusing; electrophoresis such as isoelectricfocusing; and the like which may be used alone or in combination.

When the secretory HB-EGF is expressed intracellularly by forming aninclusion body, the cells are recovered, disrupted and centrifuged inthe same manner, and the inclusion body of the secretory HB-EGF arerecovered as a precipitation fraction. The recovered inclusion body ofthe protein is solubilized with a protein denaturing agent. The proteinis made into a normal three-dimensional structure by diluting ordialyzing the solubilized solution, and then a purified product of thesecretory HB-EGF is obtained by the same isolation purification methodas above.

When the secretory HB-EGF or the derivative such as a glycosylatedproduct is secreted extracellularly, the secretory HB-EGF or thederivative such as a glycosylated product can be recovered from theculture supernatant. That is, the culture is treated by a method such ascentrifugation in the same manner as above to obtain a culturesupernatant from which solids are removed, a purified product of thepolypeptide can be obtained from the culture supernatant by the sameisolation purification method as above. Also, the secretory HB-EGF usedin the present invention can be produced by a chemical synthesis method,such as Fmoc (fluorenylmethyloxycarbonyl) method or tBoc(t-butyloxycarbonyl) method. Also, it can be chemically synthesizedusing a peptide synthesizer manufactured by Advanced ChemTech,Perkin-Elmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega,PerSeptive, Shimadzu Corporation, or the like.

(2) Immunization of Animal and Preparation of Antibody-Producing Cell

A mouse, rat or hamster 3 to 20 weeks old is immunized with the antigenprepared above, and antibody-producing cells are collected from thespleen, lymph node or peripheral blood of the animal. Also, when theincrease of a sufficient titer in the above animal is recognized due tolow immunogenecity, an HB-EGF knockout mouse may by used as an animal tobe immunized.

The immunization is carried out by administering the antigen to theanimal through subcutaneous, intravenous or intraperitoneal injectiontogether with an appropriate adjuvant (for example, complete Freund'sadjuvant, combination of aluminum hydroxide gel with pertussis vaccine,or the like). When the antigen is a partial peptide, a conjugate isproduced with a carrier protein such as BSA (bovine serum albumin), KLH(keyhole limpet hemocyanin) or the like, which is used as the antigen.

The administration of the antigen is carried out 5 to 10 times every oneweek or every two weeks after the first administration. On the 3rd to7th day after each administration, a blood sample is collected from thefundus of the eye, the reactivity of the serum with the antigen istested, for example, by enzyme immunoassay [Antibodies—A LaboratoryManual (Cold Spring Harbor Laboratory (1988)] or the like. A mouse, rator hamster showing a sufficient antibody titer in their sera against theantigen used for the immunization is used as the supply source ofantibody-producing cells.

In fusion of the antibody-producing cells and myeloma cells, on the 3rdto 7th days after final administration of the antigen, tissue containingthe antibody-producing cells such as the spleen from the immunizedmouse, rat or hamster is excised to collect the antibody-producing cell.When the spleen cells are used, the spleen is cut out in an MEM medium(Nissui Pharmaceutical) and loosened by tweezers and centrifuged (at1200 rpm, for 5 minutes). Then, the supernatant is discarded and aTris-ammonium chloride buffer (pH. 7.65) is applied for 1 to 2 minutesto remove erythrocytes. After washing 3 times with the MEM medium,antibody-producing cells for fusion are provided.

(3) Preparation of Myeloma Cell

An established cell line obtained from mouse is used as myeloma cells.Examples include 8-azaguanine-resistant mouse (derived from BALB/cmouse) myeloma cell line P3-X63Ag8-U1 (P3-U1) [Current Topics inMicrobiology and Immunology, 18, 1-7 (1978)], P3-NS1/1-Ag41 (NS-1)[European J. Immunology, 6, 511-519 (1976)], SP2/0-Ag14 (SP-2) [Nature,276, 269-270 (1978)], P3-X63-Ag8653 (653) [J. Immunology, 123, 1548-1550(1979)], P3-X63-Ag8 (X63) [Nature, 256, 495-497 (1975)] and the like.These cell lines are subcultured in an 8-azaguanine medium [a medium inwhich glutamine (1.5 mM), 2-mercaptoethanol (5×10⁻⁵ M), gentamicin (10μg/ml) and fetal calf serum (FCS) are added to RPMI-1640 medium(hereinafter referred to as “normal medium”) and 8-azaguanine (15 μg/ml)is further added] and they are subcultured in the normal medium 3 or 4days before cell fusion to ensure the cell number of 2×10⁷ or more onthe day for fusion.

(4) Cell Fusion

The above-described antibody-producing cells and myeloma cells weresufficiently washed with an MEM medium or PBS (1.83 g of disodiumhydrogen phosphate, 0.21 g of potassium dihydrogen phosphate, 7.65 g ofsodium chloride, 1 liter of distilled water, pH 7.2) and mixed to give aratio of the antibody-producing cells: the myeloma cells=5 to 10:1,followed by centrifugation (1200 rpm, 5 minutes). Then, the supernatantis discarded, and precipitated cell group is sufficiently loosen. To 10⁸of the antibody-producing cells, 0.2 to 1 mL of a mixture solution of 2g of polyethylene glycol-1000 (PEG-1000), 2 mL of MEM and 0.7 mL ofdimethylsulfoxide is added under stirring at 37° C., and 1 to 2 mL ofMEM medium is added several times every one or two minutes, and MEMmedium is added to give a total amount of 50 mL. After centrifugation(900 rpm, 5 minutes), the supernatant is discarded, the cells are gentlyloosen, and the cells are gently suspended in 100 mL of HAT medium [amedium in which hypoxanthine (10⁻⁴ M), thymidine (1.5×10⁻⁵ M) andaminopterin (4×10⁻⁷ M) is added to the normal medium] by suction andsucking out using a measuring pipette. The suspension is dispensed at100 μL/well onto a 96-well culturing plate and cultured in a 5% CO₂incubator at 37° C. for 7 to 14 days.

After the culturing, a portion of the culture supernatant is sampled anda hybridoma producing a monoclonal antibody which is reactive to all ofthe cell membrane-bound HB-EGF, the secretory HB-EGF and the membranetype HB-EGF or the purified antibody is selected by binding assay asdescribed below.

Then, cloning is carried out twice by a limiting dilution method[Firstly, HT medium (HAT medium from which aminopterin is removed) isused, and secondly, the normal medium is used], and a hybridoma whichshows a stably high antibody titer is selected as the monoclonalantibody-producing hybridoma.

(5) Preparation of Monoclonal Antibody

The hybridoma cells producing an anti-HB-EGF monoclonal antibodyobtained in (4) are administered by intraperitoneal injection into 8- to10-week-old mice or nude mice treated with pristane (0.5 ml of2,6,10,14-tetramethylpentadecane (pristane) is intraperitoneallyadministered, followed by feeding for 2 weeks) at a dose of 2×10⁶ to5×10⁷ cells/animal. The hybridoma develops ascites tumor in 10 to 21days. The ascitic fluid is collected from the mice, centrifuged (at3,000 rpm, for 5 minutes) to remove solids, subjected to salting outwith 40 to 50% saturated ammonium sulfate and then precipitated bycaprylic acid, passed through a DEAE-Sepharose column, a protein Acolumn or a gel filtration column to collect an IgG or IgM fraction as apurified monoclonal antibody.

The subclass of the antibody can be determined using a subclass typingkit by enzyme immunoassay. The amount of the protein can be determinedby the Lowry method or from the absorbance at 280 nm.

(6) Binding Assay

As the antigen, a gene-introduced cell or a recombinant protein obtainedby introducing an expression vector comprising cDNA encoding HB-EGF usedin the present invention into Escherichia coli, yeast, an insect cell,an animal cell or the like according to the method in (1) or purifiedHB-EGF or a partial peptide obtained from human tissue is used. When theantigen is a partial peptide, a conjugate is prepared with a carrierprotein such as BSA (bovine serum albumin) or KLH (keyhole limpethemocyanin) and is used.

Among these antigens, a cell line in which the secretory HB-EGF andHB-EGF are bound to the cells are dispensed into a 96-well plate andsolid-phased, then immunized animal serum, a culture supernatant of ahybridoma producing a monoclonal antibody or a purified antibody isdispensed as a first antibody and reaction is carried out. After washingwith PBS or PBS-0.05% Tween well, an anti-immunoglobulin antibodylabeled with an enzyme, a chemiluminescent substance, a radioactivesubstance or the like is dispensed as a second antibody and reaction iscarried out. After washing with PBS-Tween well, reaction according tothe labeled substance in the second antibody is carried out.

According to the method as described above, the hybridoma producing themonoclonal antibody which is reactive all of the cell membrane-boundHB-EGF, the secretory HB-EGF and the membrane type HB-EGF or thepurified antibody can be selected.

Among the monoclonal antibodies obtained, the antibody having bindinginhibition activity of the secretory HB-EGF to the HB-EGF receptorincludes monoclonal antibody KM3566 produced by hybridoma cell lineKM3566, monoclonal antibody KM3567 produced by hybridoma cell lineKM3567, and a monoclonal antibody produced by hybridoma KM3579. Thehybridoma KM3579 has been deposited to International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology (Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki,Japan) as FERM BP-10491 on Jan. 24, 2006.

In addition, a monoclonal antibody which can compete with theanti-HB-EGF monoclonal antibody of the present invention for binding toHB-EGF can be obtained by adding an antibody to be examined to the abovebinding assay system and then reacting. Namely, an antibody which cancompete with the obtained monoclonal antibody for binding to HB-EGF canbe obtained by screening an antibody which inhibits the binding of themonoclonal antibody at the time when the antibody to be examined isadded.

Furthermore, an antibody which binds to an epitope which is the same asthe epitope recognized by the anti-HB-EGF monoclonal antibody of thepresent invention can be obtained by identifying the epitope of theantibody obtained in the above binding assay, and preparing a partialsynthetic peptide, a synthetic peptide mimicking the conformationalstructure of the epitope or the like, followed by immunization.

(7) Neutralizing Activity

Furthermore, whether the obtained monoclonal antibody has neutralizingactivity to the secretory HB-EGF can be confirmed by carrying out cellgrowth inhibition assay using the HB-EGF-dependent cell.

The cell used for the cell growth inhibition assay may be any cell, solong as it the cell has a receptor to which the secretory HB-EGF canbind. Examples include a cell line obtained by introducing an EGFreceptor gene into a mouse bone marrow-derived cell line 32D clone 3(ATCC No. CRL-11346) and the like.

After the obtained monoclonal antibody is allowed to react with thesecretory HB-EGF on a plate, the above-described cell line is addedthereto, followed by culturing. As a control, the above-described cellline is similarly added to a plate to which the secretory HB-EGF isadded without monoclonal antibody and neither the secretory HB-EGF normonoclonal antibody is added, followed by culturing. The cell growthinhibition ratio can be carried out by measuring the cell number on eachplate. The monoclonal antibody having high cell growth inhibition ratiocan be selected as a monoclonal antibody having neutralizing activity.

Examples of the monoclonal antibody having neutralizing activityaccording to the present invention include monoclonal antibody KM3566produced by hybridoma cell line KM3566 and monoclonal antibody KM3567produced by hybridoma cell line KM3567. The hybridoma cell lines KM3566and KM3567 have been deposited to International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology (Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, Japan) as FERM BP-10490 and FERM BP-10573 on Jan. 24, 2006and Mar. 23, 2006, respectively.

2. Preparation of Recombinant Antibody (1) Construction of Vector forExpression of Humanized Antibody

A vector for expression of recombinant antibody may be any expressionvector for animal cell, so long as a gene encoding CH and/or CL of ahuman antibody is inserted. The vector for expression of humanizedantibody can be constructed by cloning each of genes encoding CH and CLof a human antibody into an expression vector for animal cell.

The C region of a human antibody may be CH and CL of any human antibody.Examples include the C region of IgG1 subclass of H chain in a humanantibody (hereinafter referred to as “hC γ1”), the C region of κ classof L chain in a human antibody (hereinafter referred to as “hCκ”), andthe like. As the genes encoding CH and CL of a human antibody, achromosomal DNA comprising an exon and an intron or cDNA can be used,and cDNA is also used.

As the expression vector for animal cell, any expression vector can beused, so long as a gene encoding the C region of a human antibody can beinserted thereinto and expressed therein. Examples include pAGE107[Cytotechnol., 3, 133-140 (1990)], pAGE103 [J. Biochem., 101, 1307-1310(1987)], pHSG274 [Gene, 27, 223-232 (1984)], pKCR [Proceedings of theNational Academy of Sciences of the United States of America, 78,1527-1531 (1981)], pSG1βd2-4 [Cytotechnol., 4, 173-180 (1990)] and thelike. Examples of a promoter and an enhancer used for the expressionvector for animal cell include an SV40 early promoter and enhancer [J.Biochem., 101, 1307-1310 (1987)], a Moloney mouse leukemia virus LTRpromoter and enhancer [Biochem. Biophys. Res. Commun., 149, 960-968(1987)], an immunoglobulin H chain promoter [Cell, 41, 479-487 (1985)]and enhancer [Cell, 33, 717-728 (1983)], and the like.

The vector for expression of recombinant antibody may be either of atype in which the antibody H chain and L chain exist on separate vectorsor of a type in which they exist on the same vector (hereinafterreferred to as “tandem type”). In respect of easiness of construction ofa vector for expression of human chimeric antibody and humanizedantibody, easiness of introduction into animal cells, and balancebetween the expression amounts of antibody H chain and L chain in animalcells, the tandem type of the vector for expression of humanizedantibody is preferable [Journal of Immunological Methods, 167, 271-278(1994)]. Examples of the tandem type of the vector for expression ofhumanized antibody include pKANTEX93 (WO 97/10354), pEE18 [Hybridoma,17, 559-567 (1998)], and the like.

(2) Obtaining of cDNA Encoding V Region of Non-Human Animal Antibody andAnalysis of Amino Acid Sequence

cDNAs encoding VH and VL of a non-human animal antibody such as a mouseantibody can be obtained in the following manner.

mRNA is extracted from a hybridoma to synthesize a cDNA. The synthesizedcDNA is cloned into a vector such as a phage or a plasmid to obtain acDNA library. Each of a recombinant phage or recombinant plasmidcomprising a cDNA encoding VH and a recombinant phage or recombinantplasmid comprising a cDNA encoding VL is isolated from the library byusing cDNA encoding the C region or V region of a mouse antibody as theprobe. Full length nucleotide sequences of VH and VL of the mouseantibody of interest on the recombinant phage or recombinant plasmid aredetermined, and full length amino acid sequences of VH and VL arededuced from the nucleotide sequences.

As the non-human animal, any animal can be used so long as hybridomacells can be prepared from the animal, such as mouse, rat, hamster andrabbit. The methods for preparing total RNA from the hybridoma includethe guanidine thiocyanate-cesium trifluoroacetate method [Methods inEnzymology, 154, 3-28 (1987)], and the methods for preparing mRNA fromthe total RNA include the oligo (dT) immobilized cellulose column method[Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Lab. PressNew York (1989)] and the like. Examples of the kits for preparing mRNAfrom the hybridoma include Fast Track mRNA Isolation Kit (manufacturedby Invitrogen) and Quick Prep mRNA Purification Kit (manufactured byPharmacia) and the like.

The methods for synthesizing the cDNA and for preparing the cDNA libraryinclude conventional methods [Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Lab. Press New York (1989), Current Protocols inMolecular Biology, Supplement 1-34], methods using commerciallyavailable kits such as SuperScript™ Plasmid System for cDNA Synthesisand Plasmid Cloning (manufactured by GIBCO BRL) and ZAP-cDNA SynthesisKit (manufactured by Stratagene), and the like.

In preparing the cDNA library, the vector for integrating the cDNAsynthesized using the mRNA extracted from the hybridoma as a templatemay be any vector, so long as the cDNA can be integrated. Examples ofsuitable vectors include ZAP Express [Strategies, 5, 58-61 (1992)],pBluescript II SK(+) [Nucleic Acids Research, 17, 9494 (1989)], λZAP II(manufactured by Stratagene), λgt10, λgt11 [DNA Cloning: A PracticalApproach, I, 49 (1985)], Lambda BlueMid (manufactured by Clontech),λExCell, pT7T3 18U (manufactured by Pharmacia), pcD2 [Molecular &Cellular Biology, 3, 280-289 (1983)], pUC18 [Gene, 33, 103-119 (1985)]and the like.

As Escherichia coli for introducing the cDNA library constructed with aphage or plasmid vector, any Escherichia coli can be used, so long asthe cDNA library can be introduced, expressed and maintained. Examplesinclude XL1-Blue MRF' [Journal of Biotechnology, 23, 271-289 (1992)],C600 [Genetics, 59, 177-190 (1968)], Y1088, Y1090[Science, 222, 778-782(1983)], NM522 [Journal of Molecular Biology, 166, 1-19 (1983)], K802[Journal of Molecular Biology, 16, 118-133 (1966)], JM105 [Gene 38,275-276 (1985)] and the like.

The methods for selecting the cDNA clones encoding VH and VL of anon-human animal-derived antibody from the cDNA library include colonyhybridization or plaque hybridization [Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press New York (1989)] using anisotope- or fluorescence-labeled probe. It is also possible to preparethe cDNAs encoding VH and VL by preparing primers and carrying outpolymerase chain reaction (hereinafter referred to as “PCR”) [MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press NewYork (1989), Current Protocols in Molecular Biology, Supplement 1-34]using the cDNA or cDNA library as a template.

The nucleotide sequences of the cDNAs selected by the above methods canbe determined by cleaving the cDNAs with appropriate restrictionenzymes, cloning the fragments into a plasmid such as pBluescript SK(−)(manufactured by Stratagene), and then analyzing the sequences bygenerally employed nucleotide sequence analyzing methods such as thedideoxy method [Proceedings of the National Academy of Sciences of theUnited States of America, 74, 5463-5467 (1977)] or by use of nucleotidesequence analyzers such as ABI PRISM 377 DNA Sequencer (manufactured byABI).

The full length of amino acid sequences of VH and VL are deduced fromthe determined nucleotide sequences and compared with the full length ofamino acid sequences of VH and VL of a known antibody [Sequences ofProteins of Immunological Interest, US Dept. Health and Human Services(1991)], whereby it can be confirmed that the obtained cDNAs encodeamino acid sequences which completely comprise VH and VL of the antibodyincluding secretory signal sequences. The full length amino acidsequences of VH and VL of the antibody containing signal sequences arecompared with the full length amino acid sequences of VH and VL of aknown antibody [Sequences of Proteins of Immunological Interest, USDept. Health and Human Services (1991)] to thereby deduce the length andN-terminal amino acid sequence of the signal sequences, and a subgroupto which they belong can be known. Also, the amino acid sequence of eachCDR of VH and VL can be found by comparing it with the amino acidsequence of each CDR of VH and VL of a known antibody [Sequences ofProteins of Immunological Interest, US Dept. Health and Human Services(1991)].

By carrying out homology search of sequences, such as BLAST method[Journal of Molecular Biology, 215, 403-410 (1990)], using full lengthamino acid sequence of VH and VL with any databases such as SWISS-PROTor PIR-Protein, the novelty of the sequence can be studied.

(3) Construction of Human Chimeric Antibody Expression Vector

cDNAs encoding VH and VL of antibody of non-human animal are cloned intothe upstream of genes encoding CH and CL of human antibody of vector forexpression of humanized antibody into which DNAs encoding CH and CL of ahuman antibody are inserted mentioned in the above 2(1) of this item tothereby construct a human chimeric antibody expression vector. Forexample, each cDNA encoding VH and VL of antibody of non-human animal isligated to synthetic DNA comprising a nucleotide sequence of 3′-terminalof VH or VL of antibody of non-human animal and a nucleotide sequence of5′-terminal of CH or CL of human antibody and having recognitionsequence of an appropriate restriction enzyme at both ends, and clonedso that each of them is expressed in an appropriate form in the upstreamof gene encoding CH or CL of human antibody of the vector for expressionof antibody into which DNAs encoding CH and CL of a human antibody havebeen inserted mentioned in the above 2(1) of this item to construct ahuman chimeric antibody expression vector. Also, using a plasmidcomprising cDNAs encoding VH and VL of antibody of a non-human animal asthe probe, cDNA encoding VH and VL is amplified by PCR using a primerhaving a recognition sequence of an appropriate restriction enzyme atthe 5′-terminal, and each of them is cloned into the upstream of thegenes encoding CH and CL in the vector for expression of humanizedantibody described in the above 2(1) of this item so that it can beexpressed in an appropriate form to construct a human chimeric antibodyexpression vector.

(4) Construction of cDNA Encoding V Region of Humanized Antibody(CDR-Grafted Antibody)

cDNAs encoding VH or VL of a humanized antibody can be obtained asfollows. First, amino acid sequences of FR in VH or VL of a humanantibody to which amino acid sequences of CDRs in VH or VL of a targetantibody of a non-human animal are grafted are selected. Any amino acidsequences of FR in VH or VL of a human antibody can be used, so long asthey are derived from human antibody. Examples include amino acidsequences of FRs in VH or VL of human antibodies registered in databasesuch as Protein Data Bank, amino acid sequences common to subgroups ofFRs in VH or VL of human antibodies [Sequences of Proteins ofImmunological Interest, US Dept. Health and Human Services (1991)], andthe like. Among these, in order to produce a humanized antibody havingpotent activity, amino acid sequences having high homology (at least 60%or more) with an amino acid sequence of FR in VH or VL of a targetantibody of a non-human animal is preferably selected. Then, amino acidsequences of CDRs of VH or VL of the target antibody of a non-humananimal are grafted to the selected amino acid sequence of FR in VH or VLof a human antibody, respectively, to design each amino acid sequence ofVH or VL of a humanized antibody. The designed amino acid sequences areconverted to DNA sequences by considering the frequency of codon usagefound in nucleotide sequences of genes of antibodies [Sequence ofProteins of Immunological Interest, US Dept. Health and Human Services(1991)], and the DNA sequence encoding the amino acid sequence of VH orVL of a humanized antibody is designed. Based on the designed DNAsequences, several synthetic DNAs having a length of about 150nucleotides are synthesized, and PCR is carried out using them. In thiscase, in view of the reaction efficiency and the length of DNA which canbe synthesized, it is preferred that 4 synthetic DNAs are designed foreach of VH and VL.

Furthermore, it can be easily cloned into the vector for expression ofhumanized antibody constructed in the above (2)1 of this item byintroducing the recognition sequence of an appropriate restrictionenzyme to the 5′-terminal of the synthetic DNAs existing on the bothends. After PCR, each of amplified products is cloned into a plasmidsuch as pBluescript SK (−) (manufactured by Stratagene), and thenucleotide sequence is determined according to the method described inthe above 2(2) of this item to obtain a plasmid having an amino acidsequence of VH or VL of a desired humanized antibody.

(5) Modification of Amino Acid Sequence of V Region of HumanizedAntibody

It is known that when a humanized antibody is produced by simplygrafting only CDRs in VH and VL of a target antibody of a non-humananimal into FRs of VH and VL of a human antibody, its antigen-bindingactivity is lower than that of the original antibody from a non-humananimal [BIO/TECHNOLOGY, 9, 266-271 (1991)]. As the reason, it isconsidered that several amino acid residues in not only CDRs but alsoFRs directly or indirectly relate to antigen-binding activity in VH andVL of the original antibody of a non-human animal, and as a result ofgrafting of CDRs, such amino acid residues are changed to differentamino acid residues of FRs in VH and VL of a human antibody. In order tosolve the problem, in humanized antibodies, among the amino acidsequences of FRs in VH and VL of a human antibody, an amino acid residuewhich directly relates to binding to an antigen, or an amino acidresidue which indirectly relates to binding to an antigen by interactingwith an amino acid residue in CDR or by maintaining thethree-dimensional structure of an antibody is identified and modified toan amino acid residue which is found in the original antibody of anon-human animal to thereby increase the antigen binding activity whichhas been decreased [BIO/TECHNOLOGY, 9, 266-271 (1991)]. In theproduction of a humanized antibody, how to efficiently identify theamino acid residues relating to the antigen binding activity in FR ismost important, so that the three-dimensional structure of an antibodyis constructed and analyzed by X-ray crystallography [Journal ofMolecular Biology, 112, 535-542 (1977)], computer-modeling [ProteinEngineering, 7, 1501-1507 (1994)] or the like. Although the informationof the three-dimensional structure of antibodies has been useful in theproduction of a humanized antibody, no method for producing a humanCDR-grafted antibody which can be applied to any antibodies has beenestablished yet. Therefore, various attempts must be currentlynecessary, for example, several modified antibodies of each antibody areproduced and the correlation between each of the modified antibodies andits antibody binding activity is examined.

The modification of the amino acid sequence of FR in VH and VL of ahuman antibody can be accomplished using various synthetic DNA formodification according to PCR as described in the above 2(4) of thisitem. With regard to the amplified product obtained by the PCR, thenucleotide sequence is determined according to the method as describedin 2(2) of this item so that whether the objective modification has beencarried out is confirmed.

(6) Construction of Humanized Antibody Expression Vector

A humanized antibody expression vector can be constructed by cloningeach cDNA encoding VH or VL of the constructed humanized antibody asdescribed in the above 2(4) and 2(5) of this item into upstream of eachgene encoding CH or CL of the human antibody in the vector forexpression of antibody as described in the above 2(1) of this item. Forexample, when recognizing sequences of an appropriate restrictionenzymes are introduced to the 5′-terminal of synthetic DNAs positionedat both ends among synthetic DNAs used in the construction of VH or VLof the humanized antibody in the above 2(4) and 2(5) of this item,cloning can be carried out so that they are expressed in an appropriateform in the upstream of each gene encoding CH or CL of the humanantibody in the vector for expression of antibody as described in theabove 2(1) of this item.

(7) Transient Expression of Recombinant Antibody

In order to efficiently evaluate the antigen binding activity of varioushumanized antibodies produced, the humanized antibodies can be expressedtransiently using the humanized antibody expression vector as describedin the above 2(3) and 2(6) of this item or an expression vector obtainedby modifying it. Any cell can be used as a host cell to which anexpression vector is introduced, so long as the host cell can express ahumanized antibody. Generally, COS-7 cell (ATCC CRL1651) is used in viewof its high expression amount [Methods in Nucleic Acids Research, CRCPress, 283 (1991)]. Examples of the method for introducing theexpression vector into COS-7 cell include a DEAE-dextran method [Methodsin Nucleic Acids Research, CRC Press, 283 (1991)], a lipofection method[Proceedings of the National Academy of Sciences of the United States ofAmerica, 84, 7413-7417 (1987)], and the like.

After introduction of the expression vector, the expression amount andantigen binding activity of the humanized antibody in the culturesupernatant can be determined by ELISA [Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, Chapter 14 (1988); Monoclonal Antibodies:Principles and Practice, Academic Press Limited (1996)] and the like.

(8) Stable Expression of Recombinant Antibody

A transformant cell which stably expresses a humanized antibody can beobtained by introducing the humanized antibody expression vectordescribed in the above 2(3) and 2(6) of this item into an appropriatehost cell. Examples of the method for introducing the expression vectorinto a host cell include electroporation [Cytotechnology, 3, 133-140(1990)] and the like. As the host cell into which a humanized antibodyexpression vector is introduced, any cell can be used, so long as it isa host cell which can express the humanized antibody. Examples includemouse 5P2/0-Ag14 cell (ATCC CRL1581), mouse P3×63-Ag8.653 cell (ATCCCRL1580), CHO cell in which a dihydrofolate reductase gene (hereinafterreferred to as “dhfr”) is defective [Proceedings of the National Academyof Sciences of the United States of America, 77, 4216-4220 (1980)], ratYB2/3HL.P2.G11.16Ag.20 cell (ATCC CRL1662, hereinafter referred to as“YB2/0 cell”), and the like.

After introduction of the expression vector, transformants which expressa recombinant antibody stably are selected in accordance with the methoddisclosed in Japanese Published Unexamined Patent Application No.257891/90, by culturing in a medium for animal cell culture containingan agent such as G418 sulfate (hereinafter referred to as “G418”) or thelike. Examples of the medium for animal cell culture include RPMI1640medium (manufactured by Nissui Pharmaceutical), GIT medium (manufacturedby Nihon Pharmaceutical), EX-CELL302 medium (manufactured by JRH), IMDMmedium (manufactured by GIBCO BRL), Hybridoma-SFM medium (manufacturedby GIBCO BRL), media obtained by adding various additives such as FBS tothese media, and the like. The recombinant antibody can be expressed andaccumulated in a culture supernatant by culturing the selectedtransformant cell in a medium. The expression amount and antigen bindingactivity of the recombinant antibody in the culture supernatant can bemeasured by ELISA. Also, in the transformant cell, the expression amountof the recombinant antibody can be increased by using DHFR amplificationsystem or the like according to the method disclosed in JapanesePublished Unexamined Patent Application No. 257891/90.

The recombinant antibody can be purified from the culture supernatant ofthe transformant cell by using a protein A column [Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, Chapter 8 (1988);Monoclonal Antibodies: Principles and Practice, Academic Press Limited(1996)]. Also, in addition thereto, any other conventional methods usedfor protein purification can be used. For example, the humanizedantibody can be purified by a combination of gel filtration,ion-exchange chromatography, ultrafiltration and the like. The molecularweight of the H chain or the L chain of the humanized antibody or thewhole antibody molecule can be determined by polyacrylamide gelelectrophoresis (hereinafter referred to as “PAGE”) [Nature, 227,680-685 (1970)], Western blotting [Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, Chapter 12 (1988); Monoclonal Antibodies:Principles and Practice, Academic Press Limited (1996)], and the like.

(9) Evaluation of Binding Activity of Recombinant Antibody and Antigen

The binding activity of the humanized antibody to the antigen can beevaluated by ELISA as described above.

3. Preparation of Antibody Fragment

The antibody fragment can be prepared based on the antibody as describedin the above items 1 and 2 according to the genetically engineeringmethod or the protein-chemical method.

The genetically engineering method includes a method in which a geneencoding an antibody fragment of interest is constructed and expressionand purification are carried out using an appropriate host such as ananimal cell, a plant cell, an insect cell, Escherichia coli or the like.

The protein-chemical method includes a method in which partiallyspecific cleave, purification and the like are carried out using aprotease such as pepsin or papain.

The production methods of antibody fragment including Fab, F(ab′)₂,Fab′, scFv, diabody, dsFv and peptide comprising six CDRs arespecifically described.

(1) Preparation of Fab

Fab can be prepared protein-chemically by treating IgG with protease,papain. After the treatment with papain, if the original antibody is anIgG subclass having a binding property to protein A, it is possible tocollect as a uniform Fab by passing through a protein A column toseparate from IgG molecules and Fc fragments [Monoclonal Antibodies:Principles and Practice, third edition (1995)]. In the case of anantibody of an IgG subclass having no binding property to protein A, Fabcan be collected by ion-exchange chromatography at a fraction eluted inlow salt concentrations [Monoclonal Antibodies: Principles and Practice,third edition (1995)]. Also, Fab can also be often preparedgenetic-engineeringly using Escherichia coli, and can be prepared usinginsect cells, animal cells or the like. For example, DNA encoding the Vregion of the antibody mentioned in 2(2), 2(4) and (5) of this item iscloned into a vector for expression of Fab to thereby prepare an Fabexpression vector. Any vector for expression of Fab can be used, so longas DNA for Fab can be inserted and expressed. Examples include pIT 106[Science, 240, 1041-1043 (1988)] and the like. The Fab expression vectoris introduced into an appropriate Escherichia coli to thereby produceand accumulate Fab in an inclusion body or a periplasmic space. From theinclusion body, Fab having activity can be obtained by a refoldingmethod generally used for proteins and, when expressed in periplasmicspace, Fab having activity leaks out in a culture supernatant. After therefolding or from the culture supernatant, uniform Fab can be purifiedusing a column to which an antigen is bound [Antibody Engineering, APractical Guide, W.H. Freeman and Company (1992)].

(2) Preparation of F(ab′)₂

F(ab′)₂ can be prepared protein-chemically by treating IgG withprotease, pepsin. After the treatment with pepsin, it can be recoveredas uniform F(ab′)₂ by purifying operation in the same manner as Fab[Monoclonal Antibodies: Principles and Practice, third edition, AcademicPress (1995)]. It can also be prepared by a method in which Fab′mentioned in the following 3(3) is treated with a maleimide such aso-PDM or bismaleimide to form a thioether bond or by a method in whichit is treated with DTNB [5,5′-dithiobis(2-nitrobenzoic acid)] to form anS—S bond [Antibody Engineering, A Practical Approach, IRL Press (1996)].

(3) Preparation of Fab′

Fab′ can be prepared by treating F(ab′)₂ described in the above 3(2)with a reducing agent such as dithiothreitol. Also, Fab′ can be preparedby genetic-engineeringly Escherichia coli. Furthermore, it can beprepared using an insect cell, an animal cell and the like. For example,DNA encoding the V region of the antibody mentioned in 2(2), 2(4) and2(5) of this item is cloned into a vector for expression of Fab′ tothereby prepare an Fab′ expression vector. With regard to the vector forexpression of Fab′, any vector may be used, so long as DNA for Fab′ canbe inserted and expressed. Examples include pAK19 [Bio/Technology, 10,163-167 (1992)] and the like. The Fab′ expression vector is introducedinto an appropriate Escherichia coli to produce and accumulate Fab′ inan inclusion body or periplasmic space. From the inclusion body, Fab′having activity can be obtained by a refolding method which is generallyused in proteins and, when the Fab′ is expressed in periplasmic space,it can be recovered extracellularly by disrupting the cell withtreatment such as partial digestion by lysozyme, osmotic shock andsonication. After the refolding or from the disrupted cell solution,uniform Fab′ can be purified using a protein G column or the like[Antibody Engineering, A Practical Approach, IRL Press (1996)].

(4) Preparation of scFv

scFv can be prepared genetic-engineeringly using phages, Escherichiacoli, insect cells, animal cells or the like. For example, DNA encodingthe V region of the antibody mentioned in 2(2), 2(4) and 2(5) is clonedinto a vector for expression of scFv to thereby prepare an scFvexpression vector. Any vector for expression of scFv can be used, solong as DNA for scFv can be inserted and expressed. Examples includepCANTAB5E (manufactured by Pharmacia), pHFA [Human Antibodies &Hybridomas, 5, 48-56 (1994)] and the like. When the scFv expressionvector is introduced into an appropriate Escherichia coli and a helperphage is infected, a phage which expresses scFv on the phage surface ina fused form with the surface protein of the phage can be obtained.Also, scFv can be produced and accumulated in periplasmic space or aninclusion body of Escherichia coli into which the scFv expression vectoris introduced. From the inclusion body, scFv having activity can beobtained by a refolding method generally used for proteins and, whenscFv is expressed in periplasmic space, it can be recoveredextracellularly by disrupting the cell with a treatment such as partialdigestion by lysozyme, osmotic shock, sonication or the like. After therefolding or from the disrupted cell solution, uniform scFv can bepurified using cation-exchange chromatography or the like [AntibodyEngineering, A Practical Approach, IRL Press (1996)].

(5) Preparation of Diabody

Diabody can be often prepared genetic-engineeringly using Escherichiacoli, or insect cells, animal cells or the like. For example, DNA inwhich VH and VL of the antibody described in 2(2), 2(4) and 2(5) of thisitem are linked so that amino acid residues encoded by its linker are 8or less residues is prepared and cloned into a vector for expression ofdiabody to thereby prepare a diabody expression vector. Any vector forexpression of diabody can be used, so long as DNA for diabody can beinserted and expressed. Examples include pCANTAB5E (manufactured byPharmacia), pHFA [Human Antibodies Hybridomas, 5, 48 (1994)] and thelike. Diabody can be produced and accumulated in periplasmic space or aninclusion body of Escherichia coli into which the diabody expressionvector is introduced. From the inclusion body, diabody having activitycan be obtained by a refolding method generally used for proteins and,when diabody is expressed in periplasmic space, it can be recoveredextracellularly by disrupting the cell with a treatment such as partialdigestion by lysozyme, osmotic shock, sonication or the like. After therefolding or from the disrupted cell solution, uniform diabody can bepurified using cation-exchange chromatography or the like [AntibodyEngineering, A Practical Approach, IRL Press (1996)].

(6) Preparation of dsFv

dsFv can be often prepared genetic-engineeringly using Escherichia coli,or an insect cell, an animal cell or the like. Firstly, mutation isintroduced into an appropriate position of DNAs encoding VH and VL ofthe antibody mentioned in the above 2(2), 2(4) and 2(5) of this item toprepare DNAs in which an encoded amino acid residue is replaced withcysteine. Each DNA prepared is cloned into a vector for expression ofdsFv to thereby prepare an expression vector of VH and VL. Any vectorcan be used as a vector for expression of dsFv may be used, so long asDNA for dsFv can be inserted and expressed. Examples include pULI 9[Protein Engineering, 7, 697-704 (1994)] and the like. The expressionvector of VH and VL is introduced into an appropriate E. coli and dsFvis formed and accumulated in an inclusion body or periplasmic space. VHand VL are obtained from the inclusion body or periplasmic space, mixedand subjected to a refolding method generally used for proteins tothereby obtain dsFv having activity. After the refolding, it can befurther purified by ion-exchange chromatography, a gel filtration or thelike [Protein Engineering, 7, 697-704 (1994)].

(7) Preparation of Peptide Comprising Six CDRs

A peptide comprising six CDRs can be prepared by a chemical synthesismethod such as an Fmoc method or a tBoc method. Furthermore, DNAencoding a peptide comprising CDRs is prepared and the resulting DNA iscloned into an appropriate vector for expression whereby a peptidecomprising CDRs expression vector can be prepared. With regard to avector for expression, any vector may be used so long as the DNA whichencodes a peptide comprising CDRs can be inserted and expressed.Examples include pLEX (manufactured by Invitrogen), pAX4a+ (manufacturedby Invitrogen) and the like. The expression vector is introduced into anappropriate E. coli and formed and accumulated in an inclusion body orperiplasmic space. From the inclusion body or the periplasmic space, apeptide comprising CDRs is obtained and can be purified by ion-exchangechromatography, a gel filtration or the like [Protein Engineering, 7,697 (1994)].

4. Pharmaceutical and Therapeutic Agent in the Present Invention

The pharmaceutical agent comprising the monoclonal antibody of thepresent invention as an active ingredient can be used for treatingvarious diseases relating to HB-EGF.

The diseases related to HB-EGF include cancer, heart disease,arteriosclerosis and the like. The cancer includes solid cancer such asbreast cancer, hepatic cancer, pancreatic cancer, bladder cancer,ovarian cancer and ovarian germ cell tumor. Also, it includes metastaticcancer caused by continuous, hematogenous or lympocytic metastasisaccompanied with any of the solid cancers, peritoneal dissemination orthe like. Furthermore, it includes other cancers such as cancers derivedfrom hematopoietic cells (hematological cancer or blood cancer)including leukemia (acute myelocytic leukemia, T-cell leukemia, etc.),lymphoma, myeloma, and the like.

The pharmaceutical agent comprising the antibody or antibody fragment ofthe present invention as an active ingredient is preferably supplied asa pharmaceutical preparation produced by an appropriate method wellknown in the technical field of pharmaceutics, by mixing it with one ormore pharmaceutically acceptable carriers.

It is preferred to select a route of administration which is mosteffective in treatment. Examples include oral administration andparenteral administration, such as buccal, tracheal, rectal,subcutaneous, intramuscular or intravenous administration. In the caseof an antibody or peptide formulation, intravenous administration ispreferred. The dosage form includes sprays, capsules, tablets, granules,syrups, emulsions, suppositories, injections, ointments, tapes and thelike.

The pharmaceutical preparation suitable for oral administration includesemulsions, syrups, capsules, tablets, powders, granules and the like.Liquid preparations such as emulsions and syrups can be produced using,as additives, water; sugars such as sucrose, sorbitol and fructose;glycols such as polyethylene glycol and propylene glycol; oils such assesame oil, olive oil and soybean oil; antiseptics such asp-hydroxybenzoic acid esters; flavors such as strawberry flavor andpeppermint; and the like. Capsules, tablets, powders, granules and thelike can be produced using, as additives, excipients such as lactose,glucose, sucrose and mannitol; disintegrating agents such as starch andsodium alginate; lubricants such as magnesium stearate and talc; binderssuch as polyvinyl alcohol, hydroxypropylcellulose and gelatin;surfactants such as fatty acid ester; plasticizers such as glycerin; andthe like.

The pharmaceutical preparation suitable for parenteral administrationincludes injections, suppositories, sprays and the like. Injections canbe prepared using a carrier such as a salt solution, a glucose solutionor a mixture of both thereof. Suppositories can be prepared using acarrier such as cacao butter, hydrogenated fat or carboxylic acid.Sprays can be prepared using the antibody or antibody fragment as suchor using it together with a carrier which does not stimulate the buccalor airway mucous membrane of the patient and can facilitate absorptionof the compound by dispersing it as fine particles. The carrier includeslactose, glycerol and the like. Depending on the properties of theantibody and the carrier, it is possible to produce pharmaceuticalpreparations such as aerosols and dry powders. In addition, thecomponents exemplified as additives for oral preparations can also beadded to the parenteral preparations.

Although the dose or the frequency of administration varies depending onthe objective therapeutic effect, administration method, treatingperiod, age, body weight and the like, it is usually 10 μg/kg to 30mg/kg per day and per adult.

The present invention is explained below in detail based on Examples;however, Examples are illustrations of the present invention and thepresent invention is not limited thereto.

EXAMPLE 1 Preparation of Anti-HB-EGF Monoclonal Antibody (1) Preparationof Immunogen

A freeze-dried preparation of recombinant secretory human HB-EGFmanufactured by R & D System (catalogue number 259-HE/CF) was dissolvedin Dulbecco's phosphate buffer (phosphate-buffered saline: PBS) and usedas the immunogen.

(2) Immunization of Animal and Preparation of Antibody-Producing Cell

To an HB-EGF deficient mouse [obtained from Department of Cell Biology,Research Institute for Microbial Diseases, Osaka University, PNAS, Vol.100, No. 100, 3221-3226 (2003)], 25 μg of the recombinant secretoryhuman HB-EGF prepared in Example 1(1) was administered, together with 2mg of aluminum hydroxide adjuvant (Antibodies—A Laboratory Manual, ColdSpring Harbor Laboratory, p. 99, 1988) and 1×10⁹ cells of pertussisvaccine (Chiba Serum Institute). Two weeks after the administration, 25μg of the HB-EGF alone was administered once a week and 4 times as atotal. Blood was partially collected from the venous plexus of thefundus of the eye, its serum antibody titer was examined by an enzymeimmunoassay shown below, and 3 days after the final immunization, thespleen was excised from a mouse which showed sufficient antibody titer.The spleen was cut to pieces in MEM (minimum essential medium) medium(manufactured by Nissui Pharmaceutical), and the cells were unboundusing a pair of forceps and centrifuged (1,200 rpm, 5 minutes). The thusobtained precipitate fraction was treated with Tris-ammonium chloridebuffer (pH 7.6) for 1 to 2 minutes to eliminate erythrocyte. The thusobtained precipitation fraction (cell fraction) was washed three timeswith MEM and used in the cell fusion.

(3) Enzyme Immunoassay (Binding ELISA)

The recombinant human HB-EGF of Example 1(1) was dispensed into a 96well plate for ELISA (manufactured by Greiner) at 0.5 μg/ml and 50μl/well, was allowed to stand overnight at 4° C. for adsorption and wasused in the assay. After washing the plate, 1% bovine serum albumin(BSA)-PBS was added thereto at 50 μl/well and allowed to stand at roomtemperature for 1 hour to block the remaining active groups. After theplate was allowed to stand, 1% BSA-PBS was discarded, and as the primaryantibody, antiserum of a mouse to be immunized or a hybridoma culturesupernatant was dispensed at 50 μL/well into the plate and was allowedto stand for 2 hours. After washing the plate with 0.05% polyoxyethylene(20) sorbitan monolaurate [(corresponds to Tween 20, a trademark of ICI,manufactured by Wako Pure Chemical Industries)]/PBS (hereinafterreferred to “Tween-PBS”), a peroxidase-labeled rabbit anti-mouse IgGgamma chain (manufactured by Kirkegarrd & Perry Laboratories) was addedas the secondary antibody at 50 μl/well and was allowed to stand at roomtemperature for 1 hour. After washing the plate with Tween-PBS, ABTS[2,2-azinobis(3-ethylbenzothiazole-6-sulfonic acid) ammonium] substratesolution [1 mmol/l ABTS/0.1 mol/l citrate buffer (pH 4.2), 0.1% H₂O₂]was added to develop color, and the absorbance at OD 415 nm was measuredusing a plate reader (Emax; manufactured by molecular Devices).

(4) Preparation of Mouse Myeloma Cell

An 8-azaguanine-resistant mouse myeloma cell line P3X63Ag8U.1 (P3-U1:purchased from ATCC) was cultured in 10% fetal bovine serum-supplementedRPMI 1640 (manufactured by Invitrogen), and 2×10⁷ or more of cells wereensured on the day of cell fusion and were used for the cell fusion asthe parent cell line.

(5) Preparation of Hybridoma

The mouse spleen cell obtained in Example 1(2) and the myeloma cellobtained in Example 1(4) were mixed at a ratio of 10:1, followed bycentrifugation (1,200 rpm, 5 minutes). A group of cells of the thusobtained precipitation fraction were thoroughly loosened, and then,while stirring, a mixed solution of 1 g of polyethylene glycol-1000(PEG-1000), 1 ml of MEM medium and 0.35 ml of dimethyl sulfoxide wasadded at 37° C. in an amount of 0.5 ml per 10⁸ mouse spleen cells, 1 mlof MEM medium was added to the suspension several times at an intervalof 1 to 2 minutes, and then the total volume was adjusted to 50 ml byadding MEM medium. The suspension was centrifuged (900 rpm, 5 minutes),cells of the thus obtained precipitation fraction were gently loosened,and then the cells were suspended in 100 ml of HAT medium [a mediumprepared by adding HAT Media Supplement (manufactured by Invitrogen) tothe 10% fetal bovine serum-supplemented RPMI 1640 medium], gentlythrough their sucking in and sucking out by a pipette. The suspensionwas dispensed into a 96 well culture plate at 200 μl/well, followed byculturing at 37° C. for 10 to 14 days in a 5% CO₂ incubator. After theculturing, wells which responded to the recombinant human HB-EGF wereselected by examining the culture filtrates by the enzyme immunoassaydescribed in Example 1(3), and cloning by limiting dilution method fromthe cells contained therein was repeated twice to thereby establishanti-HB-EGF monoclonal antibody-producing hybridoma cell lines KM3566,KM3567 and KM3579.

(6) Purification of Monoclonal Antibody

Each of the hybridoma cell lines obtained in Example 1(5) wasintraperitoneally injected into pristine-treated 8-week-old female nudemice at a dose of 5 to 20×10⁶ cells/animal. From 10 to 21 daysthereafter, ascitic fluid (from 1 to 8 ml/animal) was collected fromeach mouse in which ascitic fluid was accumulated caused by ascitestumor of the hybridoma. The ascitic fluid was centrifuged (3,000 rpm, 5minutes) to remove solids. Purified IgG monoclonal antibodies wereobtained by purifying them according to the caprylic acid precipitationmethod (Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory,1988). Subclass of each monoclonal antibody was determined by ELISAusing a subclass typing kit. Subclass of the monoclonal antibody KM3566was IgG 1, subclass of the monoclonal antibody KM3567 was IgG1 and thatof the monoclonal antibody KM3579 was IgG2b.

EXAMPLE 2 Reactivity of Anti-HB-EGF Monoclonal Antibody for HB-EGF

(1) Reactivity with HB-EGF by Binding ELISA

This experiment was carried out in accordance with the method shown inExample 1(3). Each of purified antibodies of anti-HB-EGF monoclonalantibodies KM3566, KM3567, KM3579, a commercially available anti-HB-EGFmonoclonal antibody MAB259 (manufactured by R & D) and a negativecontrol antibody KM511 (an anti-G-CSF derivative monoclonal antibody)was diluted stepwise from 10 μg/ml by 5-fold serial dilution and used asthe primary antibody. The results are shown in FIG. 1A.

Each of the anti-HB-EGF monoclonal antibodies KM3566, KM3567, KM3579 andMAB259 reacted with the recombinant human HB-EGF and did not react withBSA.

(2) Reactivity with HB-EGF by Western Blotting

Twenty ng per lane of the recombinant human HB-EGF (manufactured by R &D) was fractionated by SDS-polyacrylamide electrophoresis, and the gelafter electrophoresis was transferred on a PVDF membrane. After blockingthe membrane with 10% BSA-PBS, each of the purified antibodies ofanti-HB-EGF monoclonal antibodies KM3566, KM3567, KM3579, MAB259 and thenegative control antibody KM511 was diluted to 1 μg/ml using 10% BSA-PBSand was allowed to react at room temperature for 2 hours. Afterthoroughly washing the membrane with Tween-PBS, a peroxidase-labeledanti-mouse immunoglobulin antibody (manufactured by Zymed Laboratory)was diluted and was allowed to react at room temperature for 1 hour. Themembrane was thoroughly washed with Tween-PBS, bands were detected usingECL™ Western blotting detection reagents (manufactured by AmershamPharmacia).

A band of approximately from 15 to 30 kilo daltons (hereinafter referredto as “kDa”) corresponding to the molecular weight of the recombinantsecretory human HB-EGF was detected from each of the anti-HB-EGFmonoclonal antibodies KM3566, KM3567, KM3579 and MAB259.

(3) Evaluation of HB-EGF-EGFR Binding Inhibition Activity of Anti-HB-EGFMonoclonal Antibodies

The HB-EGF-EGFR binding inhibition activity of anti-HB-EGF monoclonalantibodies KM3566, KM3567, KM3579 and MAB259 was examined using 32D/EGFRcell and biotin-labeled HB-EGF.

The recombinant secretory human HB-EGF was biotin-labeled in the usualway using EZ-Link Sulfo-NHS-Biotin (manufactured by Pierce).

Each of the KM3566, KM3567, KM3579 and MAB259 was diluted stepwise from10 μg/ml by 5-fold serial dilution and dispensed into a 96 well plate at50 μl/well. Thereafter, the 32D/EGFR cell was dispensed at 1×10⁴cells/50 μl/well. Furthermore, biotin-labeled HB-EGF and Alexa647-labeled streptoavidin were diluted to optimum concentrations anddispensed at 10 μl/well and 50 μl/well, respectively, and after mixing,the mixture was allowed to react at room temperature for 3 hours undershade. A wavelength of 650 nm to 685 nm excited by a laser radiation 633nm He/Ne was measured using 8200 cellular Detection System (manufacturedby Applied Biosystems).

As a result, as shown in FIG. 1B, all of the KM3566, KM3567, KM3579 andMAB259 antibody concentration-dependently inhibited binding ofbiotinylated HB-EGF to EGFR. Accordingly, it was found that all of theanti-HB-EGF monoclonal antibodies inhibit binding of HB-EGF with EGFR.

EXAMPLE 3 Examination of Neutralization Activity of Anti-HB-EGFMonoclonal Antibodies for HB-EGF

Neutralization activity of anti-HB-EGF monoclonal antibodies KM3566,KM3567, KM3579 and MAB259 was examined by a cell growth inhibition assaywhich used an HB-EGF-dependent cell. The 32D/EGFR was used as theHB-EGF-dependent cell. Each of purified antibodies of the anti-HB-EGFmonoclonal antibodies KM3566, KM3567, KM3579 and MAB259 and a negativecontrol antibody KM511 was serially diluted from 20 μg/ml by 3- to4-fold dilution and dispensed into a 96 well plate at 50 μl/well. Next,0.1 μg/ml of recombinant human HB-EGF (manufactured by R & D) wasdispensed at 10 μl/well, and after mixing, the mixture was allowed toreact on ice for 2 hours. Thereafter, the 32D/EGFR cell was seeded atcell number of 1×10⁴ cells/40 μl/well, followed by culturing for 36hours. A viable cell measuring reagent SF (manufactured by NacalaiTesque) was added at 10 μl/well, and 2 hours thereafter, the absorbanceat OD 450 nm was measured using a plate reader (Emax; manufactured byMolecular Devices).

By regarding the absorbance of an only HB-EGF-added well as 0%inhibition ratio, and the absorbance of a no-HB-EGF and no-antibodyadded well as 100% inhibition ratio, and cell growth inhibition ratio ofeach well was calculated, with the results shown in FIG. 2A. As aresult, KM3566 showed HB-EGF-dependent cell growth inhibition activityfor the cell line 32D/EGFR, similar to the level of MAB259. Accordingly,it was found that the two antibodies have similar level of HB-EGFneutralization activity. On the other hand, since KM3579 did not inhibitgrowth of the cell line 32D/EGFR, it was found that it has no HB-EGFneutralization activity.

In addition, results of the examination of the neutralization activityof KM3567 carried out in the same manner are shown in FIG. 2B. As aresult, although its activity was weak in comparison with KM3566, KM3567had the activity to inhibit HB-EGF-dependent cell growth.

EXAMPLE 4 Examination of Reactivity of Anti-HB-EGF Monoclonal Antibodiesfor Membrane Type HB-EGF

Each of the anti-HB-EGF monoclonal antibodies KM3566, KM3567, KM3579,MAB259 and the negative control antibody KM511 was diluted with 0.1%BSA-PBS to various concentrations and added to 1 to 5×10⁵ cells of ahuman gastric cancer cell line MKN-28 (HSRRB JCRB 0253), a human ovariancancer cell line ES-2 (ATCC CRL-1978) or a human breast cancer cell lineMDA-MB-231 (ATCC HTB-26), and after mixing, the total volume wasadjusted to 50 μl. Each of these cell suspensions was allowed to reacton ice for 40 minutes and then washed three times with 0.1% BSA-PBS. Tothe cells, 50 μl of an FITC-labeled goat anti-mouse IgG+IgM (H+L)polyclonal antibody (manufactured by Kirkegaard & Perry Laboratories)prepared by diluting with 0.1% BSA-PBS was added and the mixture wasallowed to react on ice for 40 minutes. The cells were washed with 0.1%BSA-PBS and then suspended in 0.1% BSA-PBS, and the fluorescenceintensity was measured using a flow cytometer (manufactured by Coulter).

FIG. 3 shows the mean fluorescence intensity (MFI value), when each ofthe above-mentioned monoclonal antibodies was diluted 2-fold from 20μg/ml and allowed to react with MKN-28 and ES-2. FIG. 4 shows ahistogram of each antibody reactivity, when 20 μg/ml of each of theabove-mentioned monoclonal antibodies was allowed to react with thehuman breast cancer cell line MDA-MB-231. As a result, regarding MKN-28,binding activity of KM3566 and KM3579 was found. Also, in the case ofES-2, the binding activity was found in order of KM3566>KM3579.Furthermore, in the case of MDA-MB-231, the binding activity was foundin order of KM3566>KM3567 KM3579. In addition, in all of cells, the MFIvalue of MAB259 was similar to the negative control antibody KM511 andantibody-not-added negative control, and MAB259 hardly bound to cells.From the above result, it was found that the monoclonal antibodiesKM3566, KM3567 and KM3579 bind to the membrane type and cellmembrane-bound HB-EGF of cancer cell lines.

EXAMPLE 5 Isolation and Analysis of cDNA Encoding Variable Region ofAnti-HB-EGF Monoclonal Antibody

(1) Preparation of mRNA from Anti-HB-EGF Monoclonal Antibody-ProducingHybridoma Cell

About 4.8 μg of mRNA was prepared from 5×10⁷ of hybridoma cells of thehybridoma KM3566 described in Example 1, using RNAeasy Maxi kit(manufactured by QIAGEN) and Oligotex™-dt30 <Super> mRNA PurificationKit (manufactured by Takara) and in accordance with the instructionsattached thereto.

(2) DNA Cloning of H Chain and L Chain Variable Regions of Anti-HB-EGFMonoclonal Antibody KM3566

Using BD SMART™ RACE cDNA Amplification Kit (manufactured by BDBiosciences) in accordance with the instructions attached thereto, acDNA having the BD SMART II™ A Oligonucleotide sequence attached to thekit, on the 5′ terminal, was obtained from 1 μg of the mRNA of theanti-HB-EGF monoclonal antibody KM3566 obtained in Example 5(1). Usingthis cDNA as the template, and using the universal primer Amix attachedto the kit and a mouse Ig(γ)-specific primer having the nucleotidesequence represented by SEQ ID NO:6, PCR was carried out to amplify acDNA fragment of VH. Also, a cDNA fragment of VL was amplified bycarrying out PCR using a mouse Ig(κ)-specific primer having thenucleotide sequence represented by SEQ ID NO:7 instead of theIg(γ)-specific primer. After heating at 94° C. for 5 minutes, the PCRwas carried out by 5 cycles consisting of reactions at 94° C. for 30seconds and at 72° C. for 3 minutes, 5 cycles consisting of reactions at94° C. for 30 seconds, at 70° C. for 30 seconds and at 72° C. for 3minutes, and 30 cycles consisting of reactions at 94° C. for 30 seconds,at 68° C. for 30 seconds and at 72° C. for 3 minutes, followed byreaction at 72° C. for 10 seconds. The PCR was carried out using PTC-200DNA Engine (manufactured by Bio-Rad).

In order to clone the thus obtained PCR products and determine theirnucleotide sequences, they were separated by agarose gelelectrophoresis, and the PCR products of H chain and L chain, eachhaving about 600 bp, were extracted using Gel Extraction Kit(manufactured by QIAGEN). Each of the thus obtained extraction fragmentswas ligated to a SmaI-digested pBluescript II SK(−) vector usingLigation High (manufactured by TOYOBO), and then an Escherichia colistrain DH5a was transformed by the method of Cohen et al. [Proc. Natl.Acad. Sci. USA, 69, 2110 (1972)]. Plasmids were extracted from theobtained transformants using an automatic plasmid extraction device(manufactured by KURABO) and were allowed to react using BigDyeTerminator Cycle Sequencing FS Ready Reaction Kit (manufactured by PEBiosystems) in accordance with the instructions attached thereto, andthen nucleotide sequences of the cloned PCR products were analyzed by asequencer ABI PRISM 3700 of the same company. As a result, a plasmidKM3566VH10G2 containing complete length H chain cDNA and a plasmidKM3566VL10K2 containing L chain cDNA, wherein ATG sequence considered tobe the initiation codon is present in the cDNA 5′ terminal, wereobtained.

(3) Analysis of Amino Acid Sequences of Anti-HB-EGF Monoclonal AntibodyV Region

Complete nucleotide sequence of VH contained in the plasmid KM3566VH10G2is shown in SEQ ID NO:8, and complete amino acid sequence of VHcontaining a signal sequence, deduced from the sequence, in SEQ ID NO:9,complete nucleotide sequence of VL contained in the plasmid KM3566VL10K2in SEQ ID NO:10, and complete amino acid sequence of VL containing asignal sequence, deduced from the sequence, in SEQ ID NO:11. Based onthe comparison with known sequence data of mouse antibodies [SEQUENCESof Proteins of Immunological Interest, US Dept. Health and HumanServices (1991)] and the comparison with the results of analysis ofN-terminal amino acid sequences of H chain and L chain of purifiedanti-HB-EGF monoclonal antibody KM3566 using a protein sequencer(manufactured by Shimadzu Corp.: PPSQ-10), it was found that theisolated respective cDNA is a complete length cDNA encoding theanti-HB-EGF monoclonal antibody KM3566 containing a secretion signalsequence, and the amino acid sequence at positions 1 to 19 in the aminoacid sequence represented by SEQ ID NO:9 is the secretion signalsequence regarding the H chain and the amino acid sequence at positions1 to 20 in the amino acid sequence represented by SEQ ID NO:11 is thesecretion signal sequence regarding the L chain.

Next, the novelty of the VH and VL of anti-HB-EGF monoclonal antibodyKM3566 was examined. Using GCG Package (version 9.1, manufactured byGenetic Computer Group) as a sequence analyzing system, existing proteinamino acid sequence data base was retrieved by the BLASTP method[Nucleic Acids Res., 25, 3389 (1997)]. As a result, completelycoinciding amino acid sequences were not found for the VH and VL, thusconfirming that the VH and VL of anti-HB-EGF monoclonal antibody KM3566have novel amino acid sequences.

In addition, CDRs of the VH and VL of anti-HB-EGF monoclonal antibodyKM3566 were identified by comparing with amino acid sequences of knownantibodies. Amino acid sequences of CDR1, CDR2 and CDR3 of the VH ofanti-HB-EGF monoclonal antibody KM3566 are shown in SEQ ID NOs:12, 13and 14, respectively, and amino acid sequences of CDR1, CDR2 and CDR3 ofthe VL are shown in SEQ ID NOs:15, 16 and 17, respectively.

EXAMPLE 6 Preparation of Anti-HB-EGF Chimeric Antibody

(1) Construction of Anti-HB-EGF Chimeric Antibody Expression VectorpKANTEX3566

Using the humanized antibody expression vector pKANTEX93 described in WO97/10354 and the plasmids KM3566VH10G2 and KM3566VL10K2 obtained inExample 5(2), an anti-HB-EGF chimeric antibody expression vectorpKANTEX3566 was constructed in the following manner.

Using 100 ng of the plasmid KM3566VH10G2 as the template, 100 μl intotal volume of a solution consisting of 10 μl of 10×KOD buffer, 10 μlof 2 mmol/l dNTP, 2 μl of 25 mmol/1 magnesium chloride, 1 μl of each of10 μmol/l of primers having the nucleotide sequences described in SEQ IDNOs:18 and 19 and 1 μl of KOD polymerase (manufactured by TOYOBO) washeated at 96° C. for 3 minutes, followed by 25 cycles consisting ofreactions at 94° C. for 1 minute, at 55° C. for 1 minute and at 72° C.for 1 minute, further followed by reaction at 72° C. for 8 minutes. Bythis reaction, a cDNA encoding the VH of KM3566 comprising a restrictionenzyme recognizing sequence for inserting into pKANTEX93 wassynthesized. In the same manner, 100 μl in total volume of a solutionconsisting of 100 ng of the plasmid KM3566VL10K2 as the template, 10 μlof 10×KOD buffer, 10 μl of 2 mmol/l dNTP, 2 μl of 25 mmol/1 magnesiumchloride, 1 μl of each of 10 μmol/l of primers having the nucleotidesequences represented by SEQ ID NOs:20 and 21 and 1 μl of KOD polymerase(manufactured by TOYOBO) was heated at 96° C. for 3 minutes, followed by25 cycles consisting of reactions at 94° C. for 1 minute, at 55° C. for1 minute and at 72° C. for 1 minute, further followed by reaction at 72°C. for 8 minutes. By this reaction, a cDNA encoding the VL of KM3566comprising a restriction enzyme recognizing sequence for inserting intopKANTEX93 was synthesized. By purifying and concentrating each of thePCR products by ethanol precipitation and cloning it into theSmaI-digested pBluescript II SK(−), a plasmid pKM3566VH containing anucleotide sequence encoding the VH of KM3566 and a plasmid pKM3566VLcontaining a nucleotide sequence encoding the VL of KM3566 wereobtained.

Next, a restriction enzyme BsiWI (manufactured by New England Biolabs)was added to each of the vector pKANTEX93 and the pKM3566VL obtained inthe above, followed by reaction at 55° C. for 1 hour, and then arestriction enzyme EcoRI (manufactured by Takara) was added thereto,followed by reaction at 37° C. for 1 hour. This reaction solution wassubjected to agarose gel electrophoresis, and then each of a pKANTEX93EcoRI-BsiWI fragment of about 12.8 kb and a VL EcoRI-BsiWI fragment ofabout 0.43 kb was recovered using QIAquick Gel Extraction Kit(manufactured by QIAGEN). The thus obtained two fragments were ligatedusing Ligation High (manufactured by TOYOBO) in accordance with theinstructions attached thereto, and E. coli DH5α (manufactured by TOYOBO)was transformed using the thus obtained recombinant plasmid DNAsolution. By preparing each plasmid DNA from clones of transformant andconfirming it by restriction enzyme treatment, a plasmid pKANTEX3566VLcomprising the intended EcoRI-BsiWI fragment of about 0.43 kb wasobtained.

Next, a restriction enzyme ApaI (manufactured by Takara) was added toeach of the pKANTEX3566VL and pKM3566VH obtained in the above, followedby reaction at 37° C. for 1 hour, and then a restriction enzyme NotI(manufactured by New England Biolabs) was further added thereto,followed by reaction at 37° C. for 1 hour. This reaction solution wasfractionated by agarose gel electrophoresis to recover ApaI-NotIfragments, a pKANTEX3566VL of about 13.2 kb and a VH of about 0.47 kb,were respectively recovered. The thus obtained two fragments wereligated using Ligation High (manufactured by TOYOBO) in accordance withthe instructions attached thereto, and E. coli DH5α (manufactured byTOYOBO) was transformed using the thus obtained recombinant plasmid DNAsolution. By preparing each plasmid DNA from clones of transformant andconfirming it by restriction enzyme treatment, a plasmid pKANTEX3566comprising the intended ApaI-NotI fragment of about 0.47 kb wasobtained. Regarding the plasmid, after the reaction using BigDyeTerminator Cycle Sequencing FS Ready Reaction Kit (manufactured by PEBiosystems) in accordance with the instructions attached thereto, thenucleotide sequence was analyzed by a sequencer ABI PRISM 3700 of thesame company. As a result, an anti-HB-EGF chimeric antibody expressionvector pKANTEX3566 cloned with the intended cDNA encoding the VH ofKM3566 and cDNA encoding its VL was obtained. Schematic illustration ofthe vector structure is shown in FIG. 5.

(2) Expression of Anti-HB-EGF Chimeric Antibody in Animal Cell

Using the anti-HB-EGF chimeric antibody expression vector pKANTEX3566obtained in the above-mentioned (1), the anti-HB-EGF chimeric antibodywas expressed in an animal cell by a usual method [Antibody Engineering,A Practical Guide, W.H. Freeman and Company (1992)] to obtain ananti-HB-EGF chimeric antibody-producing transformant KM3966.

(3) Preparation of Purified Chimeric Antibody

After culturing the transformant KM3966 obtained in the above-mentioned(2) by a general culturing method, the cell suspension was recovered andcentrifuged at 3,000 rpm and 4° C. for 10 minutes, and then the thusrecovered supernatant was sterilized by filtration through Millex GVfilter of 0.22 μm in pore size (manufactured by Millipore). Theanti-HB-EGF chimeric antibody KM3966 was purified from the thus obtainedculture supernatant using a Protein A High-capacity resin (manufacturedby Millipore) column in accordance with the instructions attachedthereto. The purity and molecular weight were confirmed by SDS-PAGEusing a gradient gel (manufactured by ATTO, E-T520L) in accordance withthe instructions attached thereto.

The results are shown in FIG. 6. Regarding molecular weight of thepurified anti-HB-EGF chimeric antibody KM3966, a single band at around150 to 200 kDa was found under non-reducing conditions, and two bands ofabout 50 kDa and about 25 kDa under reducing conditions. These molecularweights coincide with the report stating that an IgG class antibody hasa molecular weight of about 150 kDa under non-reducing conditions but isdegraded into H chain having a molecular weight of about 50 kDa and Lchain having a molecular weight of about 25 kDa due to cutting of theintramolecular S—S bond [Antibodies—A Laboratory Manual, Cold SpringHarbor Laboratory, Chapter 14 (1988), Monoclonal Antibodies—Principalsand Practice, Academic Press Limited (1996)]. Thus, it was confirmedthat the anti-HB-EGF chimeric antibody KM3966 is expressed as anantibody molecule having correct structure.

EXAMPLE 7 Activity Evaluation of Anti-HB-EGF Chimeric Antibody (1)Binding Activity for Human Solid Cancer Cell Line

In order to evaluate binding activity of the anti-HB-EGF chimericantibody KM3966 obtained in Example 6, it was examined by fluorescentantibody technique in the following manner.

Each cell line of human ovarian cancer cell lines MCAS (JCRB 0240),RMG-I (JCRB IF 050315) and ES-2 (CRL 1978), human breast cancer celllines MDA-MB-231 (ATCC HTB-26), T47D (HTB-133), SK-BR-3 (ATCC HTB-30)and ZR-75-1 (ATCC CRL-1500) and a human gastric cancer cell line MKN-28(HSRRB JCRB 0253) was peeled off with 0.02%-EDTA Solution (manufacturedby Nacalai Tesque) and washed with PBS and then dispensed into a 96 wellU-bottom plate (manufactured by FALCON) at 1 to 2×10⁵ cells/50 μl/well.An anti-HB-EGF chimeric antibody KM3966 solution prepared to 20 μg/mlwith 1% BSA-PBS was dispensed at 50 μl/well, followed by stirred using aplate mixer, and the plate was allowed to stand on ice for 30 minutes.After washing twice with PBS, a 100 times-diluted secondary antibodyFITC-conjugated AffinityPure F(ab′)₂ Fragment Rabbit Anti-Human IgG(H+L) (manufactured by Jackson Laboratories) was added thereto at 50μl/well, followed by stirring using a plate mixer, and the plate wasallowed to stand on ice for 30 minutes under shade. After washing twicewith PBS, the fluorescence intensity was measured using a flow cytometerEPICS XL System II v. 3.0 (manufactured by BECKMAN COULTER). As thenegative control antibody, anti-FGF-8 chimeric antibody KM3034 (US2004-0253234) was used.

The results are shown in FIG. 7. The anti-HB-EGF chimeric antibodyKM3966 bound to the membrane type and cell membrane-bound HB-EGF of allof the human solid cancer cell lines.

(2) Measurement of Binding Activity of Anti-HB-EGF Chimeric AntibodyKM3966 for Human HB-EGF

In order to analyze binding activities of a mouse antibody KM3566 andthe chimeric antibody KM3966 for human HB-EGF in reaction kinetics, thebinding activity measurement was carried out using Biacore. All of thefollowing operations were carried out using Biacore T-100 (manufacturedby Biacore). Human HB-EGF (manufactured by R & D) prepared into 5 μg/mlusing HBS-EP Buffer (manufactured by Biacore) was immobilized onto a CM5 sensor chip (manufactured by Biacore) to a level of 80 RU (resonanceunit) by an amine coupling method. Thereafter, each antibody diluted5-fold from 9 nmol/l was let flow on the chip at a rate of 10 μl/min,and by analyzing the sensor-gram at each concentration, the associationrate constant and dissociation rate constant of each antibody for humanHB-EGF were calculated.

As a result, it was found that dissociation reaction is hardly foundafter antibody binding with human HB-EGF within the antibodyconcentration range in the case of both antibodies, so that thedissociation rate constant could not be calculated. On the other hand,the association rate constant could be calculated, with the resultsshown in Table 1. It was confirmed based on the results that bothantibodies almost the same binding activity for human HB-EGF.

TABLE 1 Antibody Ka (1/Ms) KM3566 2.7 × 10⁵ KM3966 2.4 × 10⁵

(3) Reactivity of Anti-HB-EGF Monoclonal Antibody for CellMembrane-Bound HB-EGF

Each cell line was peeled off with 0.02%-EDTA Solution (manufactured byNacalai Tesque) and washed with PBS, followed by mixing with RPMI 1640medium (manufactured by GIBCO-BRL) and centrifugation at 300 G for 5minutes, and the supernatant was discarded. To the cells, 1 μg/ml ofrecombinant human HB-EGF (manufactured by R & D) diluted with 0.1%BSA-PBS was added and was allowed to react at 37° C. for 10 minutes.When the recombinant human HB-EGF was not added, 0.1% BSA-PBS alone wasadded and was allowed to react at 37° C. for 10 minutes in the samemanner. After washing twice with 1% BSA-PBS, an anti-HB-EGF chimericantibody KM3966 solution prepared into 10 μg/ml using 1% BSA-PBS wasdispensed at 50 μl/well, followed by stirring using a plate mixer, andthe plate was allowed to stand on ice for 30 minutes. After washingtwice with PBS, a secondary antibody FITC-conjugated AffinityPureF(ab′)₂ Fragment Rabbit Anti-Human IgG (H+L) (manufactured by JacksonLaboratories) diluted 100-fold was added thereto at 50 μl/well, followedby stirring using a plate mixer, and the plate was allowed to stand onice for 30 minutes under shade. After washing twice with PBS, thefluorescence intensity was measured using a flow cytometer EPICS XLSystem II v. 3.0 (manufactured by BECKMAN COULTER). As the negativecontrol antibody, an anti-FGF 8 chimeric antibody (US 2004-0253234) wasused.

As a result, in all of the cell lines, reactivity of the anti-HB-EGFchimeric antibody KM3966 was increased in the cells treated withrecombinant HB-EGF in comparison with the untreated cells (FIG. 8).Accordingly, it was found that the anti-HB-EGF chimeric antibody KM3966of the invention binds to both of the membrane type and cellmembrane-bound HB-EGF.

(4) Neutralization Activity for Human Solid Cancer Cell Lines

In order to evaluate HB-EGF neutralization activity of the anti-HB-EGFchimeric antibody KM3966 obtained in Example 6, HB-EGF-dependent cellgrowth inhibition activity was measured. As the HB-EGF-dependent cells,an HB-EGF-positive human ovarian cancer cell line RMG-I (JCRB IF 050315)and a human gastric cancer cell line MKN-28 (HSRRB JCRB) were used.

Each cell line was peeled off with 0.02%-EDTA Solution (manufactured byNacalai Tesque) and washed with PBS, followed by mixing with RPMI 1640medium (manufactured by GIBCO-BRL) (serum-free) and centrifugation at300 G for 5 minutes, and the supernatant was discarded. After suspendingthe cells in the same medium, RMG-I and MKN-28 were seeded into a 96well plate at 2.5×10³ cells/50 μl/well and at 1×10⁴ cells/50 μl/well,respectively. The recombinant human HB-EGF (manufactured by R & D)diluted with 0.1% BSA-PBS was added at 50 μl/well having a concentrationof 3 ng/ml in the case of RMG-I, or at 50 μl/well portions having aconcentration of 30 ng/ml in the case of MKN-28, and then anti-HB-EGFchimeric antibody KM3966 was diluted 10-fold by 4 serial steps startingfrom 30 μg/ml, and was added at 50 μl/well portions, followed by mixing.As the negative control antibody, a human IgG (manufactured byMitsubishi Pharma Corp.) was used. After culturing at 37° C. for 72hours, a viable cell measuring reagent WST-1 (manufactured by NacalaiTesque) was added at 15 μl/well, and 2 hours thereafter, the absorbanceat OD 450 nm was measured using a plate reader (Emax; manufactured byMolecular Devices).

The results are shown in FIG. 9. Both of the RMG-I and MKN-28 showedcell growth caused by the addition of HB-EGF and showed HB-EGF-dependentcell growth. The anti-HB-EGF chimeric antibody KM3966 suppressed theHB-EGF-dependent cell growth in antibody concentration-dependent manner,thus showing neutralization activity.

(5) Antibody-Dependent Cellular Cytotoxicity (ADCC Activity)

ADCC activity of the anti-HB-EGF chimeric antibody KM3966 obtained inExample 6 was measured in accordance with the method shown below.

(5)-1 Preparation of Target Cell Solution

Each of the human ovarian cancer cell lines MCAS, RMG-I and ES-2, humanbreast cancer cell lines MDA-MB-231, T47D, SK-BR-3 an ZR-75-1 and ahuman gastric cancer cell stain MKN-28 was peeled off with 0.02%-EDTASolution (manufactured by Nacalai Tesque), washed with RPMI 1640 medium(manufactured by Invitrogen) containing 1% FCS (manufactured by JRH) andnot containing phenol red (to be referred to as ADCC medium hereinafter)and then adjusted to an optimum concentration using the same medium tobe used as a target cell solution.

(5)-2 Preparation of Effector Cell Solution

Peripheral blood mononuclear cell (PBMC) was separated from peripheralblood of a healthy person by the method shown below. From a healthyperson, 50 ml of peripheral blood was collected using a syringecontaining a small amount of heparin sodium injection N “Shimizu”(manufactured by Shimizu Pharmaceutical). The thus collected peripheralblood was diluted by adding the same volume of physiological saline (themanufacture's name), followed by thoroughly stirring. Polymorphprep(manufactured by NYCOMED) was dispensed into 15 ml capacity tubes(manufactured by Greiner) at about 6.5 ml, and the same volume of thediluted peripheral blood was gently overlaid thereon and centrifuged at800 G for 30 minutes at room temperature to separate the mononuclearcell layer. After washing twice using the ADCC medium, it was preparedto the optimum concentration with the same medium and used as theeffector cell solution.

(5)-3 Measurement of ADCC Activity

An antibody dilution solution was dispensed in 50 μl portions into wellsof a 96 well U-bottom plate, 50 μl of the target cell solution preparedin (4)-1 and 50 μl of the effector cell solution prepared in (4)-2 wereadded thereto (ratio of the effector cell (E) and the target cell (T)was set to 25), the total volume was adjusted to 150 μl, and thereaction was carried out at 37° C. for 4 hours. The value of target cellspontaneous release was obtained by adding 50 μl of the target cellsolution and 100 μl of the medium, and the value of target cell andeffector cell spontaneous release by adding 50 μl of the target cellsolution, 50 μl of the effector cell solution and 50 μl of the medium.The value of target cell total release was obtained by adding 50 μl ofthe target cell solution and 80 μl of the medium, and by adding 20 μl of9% Triton X-100 solution 45 minutes before completion of the reaction.After the reaction, the plate was centrifuged, and lactate dehydrogenase(LDH) activity in the supernatant was detected by measuring theabsorbance using LDH-Cytotoxic Test (manufactured by Wako) in accordancewith the instructions attached thereto. The ADCC activity was calculatedby the following formula.

ADCC activity (%)=([absorbance of sample]−[absorbance of target cell andeffector cell spontaneous release])/([absorbance of target cell totalrelease]−[absorbance of target cell spontaneous release])×100  (Formula)

The results are shown in FIG. 10. The anti-HB-EGF chimeric antibodyKM3966 showed antibody-dependent cytotoxicity for the HB-EGF-positivehuman solid cancer cell lines.

(6) Evaluation of Antitumor Activity Using Mouse Xenograft

In order to evaluate antitumor activity of the anti-HB-EGF chimericantibody KM3966 obtained in Example 6, evaluation was carried out usingmouse xenograft early cancer model and advanced cancer models of humanovarian cancer and human breast cancer.

(6)-1 Evaluation by Early Cancer Model

Each of human ovarian cancer cell lines MCAS and ES-2 was detached with0.02%-EDTA Solution (manufactured by Nacalai Tesque) and was washed withPBS, and RPMI 1640 medium (manufactured by GIBCO-BRL) was added thereto,followed by centrifugation at 300 G for 5 minutes to discard thesupernatant. The cells were washed by a centrifugation operation byadding the same medium and then prepared to the optimum density, and 100μl of the thus prepared cell suspension was subcutaneously transplantedinto the right frank of chest of each 8-week-old female SCID mouse(manufactured by CLEA Japan). Starting on the same day, 100 μl of anantibody solution diluted with PBS was administered from the caudal veinin the case of the antibody administration group, or PBS alone in thecase of the control group (5 to 7 animals per group). The administrationwas carried out twice a week, 8 times in total, and the tumor diameterwas measured using slide calipers starting at the time when a tumor wasfound. The tumor volume was calculated by the following formula.

Tumor volume (mm³)=length×breadth²×0.5  (Formula)

The results are shown in FIG. 11. The anti-HB-EGF chimeric antibodyKM3966 significantly inhibited tumor growth of the ovarian cancer celllines MCAS and ES-2. Accordingly, it was found that the anti-HB-EGFchimeric antibody KM3966 has antitumor effect in the early cancer model.

(6)-2 Evaluation by Advanced Cancer Model

Each of human ovarian cancer cell lines MCAS and ES-2 and a human breastcancer cell line MDA-MB-231 was detached with 0.02%-EDTA Solution(manufactured by Nacalai Tesque) and washed with PBS, and then RPMI 1640medium (manufactured by GIBCO-BRL) was added thereto, followed bycentrifugation at 300 G for 5 minutes to discard the supernatant. Afterthe same medium was added, the cells were washed by centrifugation andthen were prepared to the optimum density, and 100 μl of the thusprepared cell suspension was subcutaneously transplanted into the rightarmpit of each 6-to 8-week-old female SCID mouse (manufactured by CLEAJapan). By observing the development, the mice were selected when thetumor volume became about 100 mm³, and their grouping was carried out insuch a manner that the average tumor volume in respective groups becamesimilar level. Starting on the same day, 100 μl of an antibody solutiondiluted with PBS was administered from the caudal vein in the case ofthe antibody administration group, or PBS alone in the case of thecontrol group (6 or 7 animals per group). The administration was carriedout twice a week, 8 times in total, and the tumor diameter was measuredusing slide calipers starting at the time of the antibodyadministration. The tumor volume was calculated by the followingformula.

Tumor volume (mm³)=length×breadth²×0.5  (Formula)

The results are shown in FIG. 12. As a result, the anti-HB-EGF chimericantibody KM3966 significantly inhibited tumor growth of the ovariancancer cell lines MCAS and ES-2 and breast cancer cell line MDA-MB 231.Accordingly, it was found that the anti-HB-EGF chimeric antibody KM3966has the antitumor activity in the advanced cancer model.

EXAMPLE 8 Evaluation of Reactivity and Antibody-Dependent CellularCytotoxicity (ADCC Activity) of Anti-HB-EGF Antibody for Human BloodCancer Cell Lines (1) HB-EGF Expression Analysis in Human Blood CancerCell Lines

In order to evaluate HB-EGF expression in human blood cancer cell lines,it was examined by the fluorescent antibody technique. Each of humanacute myelogenous leukemia cell lines ML-1 (DSMZ ACC 464), MOLM-13 (DSMZACC 554), MV-4-11 (ATCC CRL 9591), HL-60 (ATCC CRL-240), NB-4 (DSMZ ACC207) and KG-1a (ATCC CCL-246.1) and human T cell leukemia cell linesKarpas 299 (DSMZ ACC 31) and Jurkat (RCB RCB 0806) was washed with PBS,was prepared to the optimum density and then was dispensed into a 96well U-bottom plate (manufactured by FALCON) at 50 μl/well (about 2×10⁵cells). An anti-HB-EGF mouse antibody KM3566 solution prepared into 20μg/ml with 1% BSA-PBS was dispensed at 50 μl/well, followed by stirringusing a plate mixer, and the plate was allowed to stand on ice for 30minutes. After washing twice with PBS, a 50-fold diluted secondaryantibody Anti-mouse Igs/FITC Goat F(ab′)₂ (manufactured by DAKO) wasadded thereto at 50 μl/well, followed by stirring using a plate mixer,and the plate was allowed to stand on ice for 30 minutes under shade.After washing twice with PBS, the fluorescence intensity was measuredusing a flow cytometer EPICS XL System II v. 3.0 (manufactured byBECKMAN COULTER). As the negative control antibody, mouse IgG1(manufactured by DAKO) was used.

The results are shown in FIG. 13. The KM3566 specifically bound to the Tcell leukemia and acute myelogenous leukemia cell lines. Accordingly, itwas confirmed that HB-EGF is expressing in human blood cancer celllines.

(2) Antibody-Dependent Cellular Cytotoxicity (ADCC Activity) ofAnti-HB-EGF Chimeric Antibody for Human Blood Cancer Cell Lines

ADCC activity of the anti-HB-EGF chimeric antibody KM3966 for the acutemyelogenous leukemia cell lines in which expression of HB-EGF wasconfirmed was measured by the method shown below.

(2)-1 Preparation of Target Cell Solution

Each of the human acute myelogenous leukemia cell lines ML-1, MOLM-13,MV-4-11, HL-60, NB-4 and KG-1a was washed with PBS, washed with the ADCCmedium and then prepared to the optimum density with the same medium andused as the target cell solution.

(2)-2 Preparation of Effector Cell Solution

Peripheral blood mononuclear cell (PBMC) was separated from peripheralblood of a healthy person by the method shown below. From a healthyperson, 50 ml of peripheral blood was collected using a syringecontaining a small amount of heparin sodium injection N “Shimizu”(manufactured by Shimizu Pharmaceutical). The thus collected peripheralblood was diluted by adding the same volume of physiological saline(manufactured by Otsuka Pharmaceutical), followed by thoroughlystirring. Polymorphprep (manufactured by NYCOMED) was dispensed into 15ml capacity tubes (manufactured by Greiner) at about 6.5 ml, and thesame volume of the diluted peripheral blood was gently overlaid thereon,followed by centrifugation at 800 G for 30 minutes at room temperatureto separate the mononuclear cell layer. After washing twice using theADCC medium, it was adjusted to the optimum concentration with the samemedium and used as the effector cell solution.

(2)-3 Measurement of ADCC Activity

An antibody dilution solution was dispensed into wells of a 96 wellU-bottom plate (manufactured by FALCON) at 50 μl, 50 μl of the targetcell solution prepared in (2)-1 and 50 μl of the effector cell solutionprepared in (2)-2 were added thereto (ratio of the effector cell (E) andthe target cell (T) was set to 25), the total volume was adjusted to 150μl, and the reaction was carried out at 37° C. for 4 hours. The value oftarget cell spontaneous release was obtained by adding 50 μl of thetarget cell solution and 100 μl of the medium, and the value of targetcell and effector cell spontaneous release by adding 50 μl of the targetcell solution, 50 μl of the effector cell solution and 50 μl of themedium. The value of target cell total release was obtained by adding 50μl of the target cell solution and 80 μl of the medium, and by adding 20μl of 9% Triton X-100 solution 45 minutes before completion of thereaction. After the reaction, the plate was centrifuged, and lactatedehydrogenase(LDH) activity in the supernatant was detected by measuringthe absorbance using LDH-Cytotoxic Test (manufactured by Wako) inaccordance with the instructions attached thereto. The ADCC activity wascalculated by the following formula.

ADCC activity (%)=([absorbance of sample]−[absorbance of target cell andeffector cell spontaneous release])/([absorbance of target cell totalrelease]−[absorbance of target cell spontaneous release])×100  (Formula)

The results are shown in FIG. 14. The anti-HB-EGF chimeric antibodyKM3966 showed antibody-dependent cytotoxicity for the HB-EGF-positivehuman blood cancer cell lines. Accordingly, it was suggested that thereis a possibility that the anti-HB-EGF monoclonal antibody andrecombinant antibody of the invention are effective not only for solidcancers such as ovarian cancer expressing HB-EGF but also for bloodcancers such as acute myelogenous leukemia and T cell leukemia.

EXAMPLE 9 Preparation of ANTI-HB-EGF Humanized Antibody (1) Designing ofAmino Acid Sequences of VH and VL of Anti-HB-EGF Humanized Antibody

Firstly, the amino acid sequence of VH of anti-HB-EGF humanized antibodywas designed as follows.

In order to graft amino acid sequences of CDRs 1 to 3 of antibody VHrepresented by SEQ ID NOs:12 to 14, respectively, an FR amino acidsequence of VH of a human antibody was selected. Kabat et al. haveclassified the VH of various known human antibodies into three subgroups(HSG I to III) based on the homology of the amino acid sequences andfurther reported consensus sequences among respective subgroups(SEQUENCES of Proteins of Immunological Interest, US Dept. Health andHuman Services, 1991). Since these consensus sequences have apossibility that the immunogenicity is reduced in human, it was decidedto design a VH amino acid sequence of an anti-HB-EGF humanized antibodybased on these consensus sequences. In order to prepare an anti-HB-EGFhumanized antibody having more higher binding activity in designing it,it was decided to select an FR amino acid sequence having the highesthomology with the FR amino acid sequence of VH of anti-HB-EGF mouseantibody KM3566, among FR amino acid sequences of consensus sequences ofthe three subgroups of VH of human antibodies.

As a result of homology retrieval, homologies of HSG I, HSG II and HSGIII were 73.6%, 50.6% and 56.3%, respectively. Thus, the amino acidsequence of FR of VH region of KM3566 had the highest homology with thesubgroup I.

Based on the above results, CDRs amino acid sequences of VH of theanti-HB-EGF mouse antibody KM3566 were grafted to an appropriateposition of the FR amino acid sequence of the consensus sequence ofsubgroup I of VH of the human antibody. However, although Lys atposition 74 in the VH amino acid sequence of KM3566 described in SEQ IDNO:9 is not the amino acid residue having the highest usage frequencycited by Kabat et al., in a corresponding region of the human antibodyFR amino acid sequence, but is an amino acid residue which is used at arelatively high frequency, so that it was decided to use theabove-mentioned amino acid residue which is found in the amino acidsequence of KM3566. Thus, the amino acid sequence HV0 of VH ofanti-HB-EGF humanized antibody, represented by SEQ ID NO:22, wasdesigned.

Next, the amino acid sequence of VL of the anti-HB-EGF humanizedantibody was designed as follows.

In order to graft amino acid sequences of CDRs 1 to 3 of antibody VLrepresented by SEQ ID NOs:15 to 17, respectively, an FR amino acidsequence of VL of a human antibody was selected. Kabat et al. haveclassified the VL of various known human antibodies into four subgroups(HSG Ito IV) based on the homology of the amino acid sequences and thenreported consensus sequences among respective subgroups [Sequences ofProteins of Immunological Interest, US Dept. Health and Human Services,(1991)]. Accordingly, similar to the case of VH, an FR amino acidsequence having the highest homology with the FR amino acid sequence ofVL of anti-HB-EGF mouse antibody KM3566 was selected from FR amino acidsequences of consensus sequences of the four subgroups of VL of humanantibodies.

As a result of homology search, homologies of HSG I, HSG II, HSG III andHSG IV were 75.0%, 75.0%, 71.3% and 81.3%, respectively. Thus, the aminoacid sequence of FR of VL region of KM3566 had the highest homology withthe subgroup IV.

Based on the above results, CDRs amino acid sequence of VL of theanti-HB-EGF mouse antibody KM3566 were grafted to an appropriateposition of the amino acid sequence of FR of the consensus sequence ofsubgroup IV of VL of the human antibody. However, although Leu atposition 110 in the VL amino acid sequence of KM3566 described in SEQ IDNO:11 is not the amino acid residue having the highest usage frequencycited by Kabat et al., in a corresponding region of the human antibodyFR amino acid sequence, but is an amino acid residue which is used at arelatively high frequency, so that it was decided to use theabove-mentioned amino acid residue which is found in the amino acidsequence of KM3566. Thus, the amino acid sequence LV0 of VL ofanti-HB-EGF humanized antibody, represented by SEQ ID NO:23, wasdesigned.

The thus designed amino acid sequence HV0 of VH and amino acid sequenceLV0 of VL of the anti-HB-EGF humanized antibody are sequences in whichonly the CDRs amino acid sequences of the anti-HB-EGF mouse antibodyKM3566 were grafted to the FR amino acid sequence of the selected humanantibody. However in general, when a humanized antibody is prepared, thebinding activity is decreased in many cases by grafting of a mouseantibody CDR amino acid sequence alone. Thus, in order to avoid thisreduction of binding activity, among FR amino acid residues differentbetween a human antibody and a mouse antibody, modification of aminoacid residues considered to have influences on the binding activity iscarried out together with grafted together with grafting of CDR aminoacid sequences. Accordingly, in this Example, the FR amino acid residuesconsidered to have influences on the binding activity was alsoidentified as follows.

Firstly, a three-dimensional structure of an antibody V region (HV0LV0)consisting of the amino acid sequence HV0 of VH and amino acid sequenceLV0 of VL of anti-HB-EGF humanized antibody designed in the above, wasconstructed by computer modeling technique. This construction wascarried out using a software AbM (manufactured by Oxford Molecular) forthe preparation of three-dimensional structure coordinates and asoftware Pro-Explore (manufactured by Oxford Molecular) or ViewerLite(manufactured by Accelrys) for the display of three-dimensionalstructure, in accordance with respective instructions attached thereto.Also, a computer model of the three-dimensional structure of the Vregion of anti-HB-EGF mouse monoclonal antibody KM3566 was alsoconstructed in the same manner. In addition, a three-dimensionalstructure model was constructed in the same manner by preparing an aminoacid sequence in which amino acid residues different from anti-HB-EGFmouse antibody KM3566 in the FR amino acid sequences of VH and VL ofHV0LV0 were selected and modified into the amino acid residues ofanti-HB-EGF mouse antibody KM3566. By comparing three-dimensionalstructures of V regions of these prepared anti-HB-EGF mouse antibodyKM3566, HV0LV0 and modified product, the amino acid residues consideredto have influences on the binding activity of antibody were identified.

As a result, as amino acid residues among the FR amino acid residues ofHV0LV0, considered to have influences on the antibody activity bychanging three-dimensional structure of the antigen-binding region, Alaat position 9, Val at position 20, Thr at position 30, Arg at position38, Pro at position 41, Met at position 48, Arg at position 67, Val atposition 68, Ile at position 70, Tyr at position 95 and Val at position118, were selected regarding HV0, and Leu at position 15, Ala atposition 19, Ile at position 21, Pro at position 49 and Leu at position84 regarding LV0, respectively. By modifying at least one or more ofthese selected amino acid residues to the amino acid residues presentingin the same region of mouse antibody KM3566, the VH and VL of humanizedantibody having various modifications were designed. Specifically,regarding the antibody VH, at least one modification was introducedamong the amino acid modifications in which, in the amino acid sequencerepresented by SEQ ID NO:22, Ala at position 9 was substituted with Thr,Val at position 20 was substituted with Leu, Thr at position 30 wassubstituted with Arg, Arg at position 38 was substituted with Lys, Proat position 41 was substituted with Thr, Met at position 48 wassubstituted with Ile, Arg at position 67 was substituted with Lys, Valat position 68 was substituted with Ala, Ile at position 70 wassubstituted with Leu, Tyr at position 95 was substituted with Phe andVal at position 118 was substituted with Leu. Also, regarding the VL, atleast one modification was introduced among the amino acid modificationsin which, in the amino acid sequence represented by SEQ ID NO:23, Leu atposition 15 was substituted with Val, Ala at position 19 was substitutedwith Val, Ile at position 21 was substituted with Met, Pro at position49 was substituted with Ser and Leu at position 84 was substituted withVal.

(2) Construction of cDNA Encoding VH of Anti-HB-EGF Humanized Antibody

A cDNA encoding the anti-HB-EGF humanized antibody VH amino acidsequence HV0 designed in this Example (1) was constructed using PCR inthe following manner.

Firstly, the designed amino acid sequence was made into completeantibody amino acid sequence by ligating with the secretion signalsequence of H chain of anti-HB-EGF mouse antibody KM3566 described inpositions 1 to 19 of SEQ ID NO:9. Next, the amino acid sequence wasconverted into genetic codons. When two or more genetic codons werepresent for one amino acid residue, corresponding genetic codon wasdetermined by taking the codon usage found in nucleotide sequences ofantibody genes into consideration [SEQUENCES of Proteins ofImmunological Interest, US Dept. Health and Human Services, 1991)]. Thenucleotide sequence of a cDNA encoding the complete antibody V regionamino acid sequence was designed by ligating the thus determined geneticcodons, and binding nucleotide sequences of primers for PCRamplification (including restriction enzyme recognition sequences forcloning into a humanized antibody expression vector) were added to the5′-terminal and 3′-terminal. The thus designed nucleotide sequence wasdivided into a total of 4 nucleotide sequences, each having about 100nucleotides, from the 5′-terminal side (adjoining nucleotide sequencesare designed such that the termini have an overlapping sequence of about20 nucleotides), and synthetic DNA fragments (SEQ ID NOs:24 to 27) weresynthesized by arranging them in alternating order of sense chain andantisense chain.

Each of the synthetic DNA fragments (SEQ ID NOs:24 to 27) was added to50 μl of a reaction solution to a final concentration of 0.1 μmol/l, andPCR was carried out using 0.5 μmol/l of T3 primer (manufactured byTakara Shuzo), 0.5 μmol/l of T7 primer (manufactured by Takara Shuzo)and 1 unit of KOD polymerase (manufactured by TOYOBO), in accordancewith the instructions attached to the KOD polymerase. As the reactionconditions in this case, the PCR was carried out in accordance with theconditions described in the instructions (30 cycles consisting ofreactions at 94° C. for 30 seconds, at 50° C. for 30 seconds and at 74°C. for 60 seconds). The reaction solution was subjected to ethanolprecipitation, and the precipitate was dissolved in sterile water,subjected to appropriate restriction enzyme treatment and then ligatedwith a plasmid pBluescript II SK(−). E. coli DH5α was transformed usingthe thus obtained recombinant plasmid DNA solution, each plasmid DNA wasprepared from each of the transformants, and then its nucleotidesequence was analyzed using Big Dye Terminator Cycle Sequencing Kit(manufactured by Applied Biosystems). As a result, a plasmid having thenucleotide sequence of interest was obtained.

Next, the FR amino acid residues designed in Example 9 (1) was modifiedby preparing a synthetic DNA having mutation and carrying out theabove-mentioned PCR, or by carrying out PCR using a plasmid DNAcontaining a cDNA encoding the HV0 prepared in the above, as thetemplate, and synthetic DNA fragments as primers, and isolating anamplified gene fragment. Gene codons of the amino acid residues aftermodification were prepared in such a manner that they became the genecodons found in the anti-HB-EGF mouse antibody KM3566. In addition,unless otherwise indicated below, the reaction was carried out by 35cycles of the PCR, each cycle consisting of reactions at 94° C. for 30seconds, at 55° C. for 30 seconds and at 72° C. for 60 seconds. The PCRwas carried out using KOD-plus polymerase (manufactured by TOYOBO).Also, the synthetic DNA used herein is a product of FASMAC Co., Ltd.

Next, the VH wherein Val²⁰Thr³⁰Met⁴⁸Val⁶⁸Ile⁷⁰Tyr⁹⁵Val¹¹⁸ of the aboveHV0 is modified to Leu²⁰Arg³⁰Ile⁴⁸Ala⁶⁸Leu⁷⁰Phe⁹⁵Leu¹¹⁸ (hereinafterreferred to as HV7) was prepared by the following method.

PCR was carried out by adding 0.1 μmol/l of synthetic DNAs having aminoacid mutation (SEQ ID NOs: 32 to 35) and 0.4 μmol/l of primers M13RVprimer and M13M4 primer positioning at both termini thereof. The PCRsolution was purified using a Gel extraction kit, 0.8 to 1.5% agaroseelectrophoresis was carried out and the intended gene fragment of about0.45 kbp was extracted using the Gel extraction kit. Sub-cloning intopBluesecript II SK(−) (hereinafter referred to as pBS) which had beentreated with a specific restriction enzyme Sinai was carried out toobtain a vector pBS/HV7 that contains a gene (SEQ ID NO:36) coding forthe VH amino acid sequence HV7 of anti-HB-EGF humanized antibody.

(3) Construction of cDNA Encoding VL of Anti-HB-EGF Humanized Antibody

A cDNA encoding the anti-HB-EGF humanized antibody VL amino acidsequence designed in this Example (1) was constructed using PCRdescribed below.

Firstly, the designed amino acid sequence was made into completeantibody amino acid sequence by ligating with the secretion signalsequence of L chain of anti-HB-EGF mouse antibody KM3566, represented bypositions 1 to 20 of SEQ ID NO:11. Next, the amino acid sequence wasconverted into genetic codons. When two or more genetic codons werepresent for one amino acid residue, corresponding genetic codon wasdetermined by taking the codon usage found in nucleotide sequences ofantibody genes into consideration [SEQUENCES of Proteins ofImmunological Interest, US Dept. Health and Human Services, 1991)]. Thenucleotide sequence of a cDNA encoding the complete antibody V regionamino acid sequence was designed by ligating the thus determined geneticcodons, and binding nucleotide sequences of primers for use in the PCRamplification (including restriction enzyme recognition sequences forcloning into a humanized antibody expression vector) were added to the5′-terminal and 3′-terminal. The thus designed nucleotide sequence wasdivided into a total of 4 nucleotide sequences, each having about 100nucleotides, from the 5′-terminal side (adjoining nucleotide sequencesare designed such that the termini have an overlapping sequence of about20 nucleotides), and synthetic DNA fragments (SEQ ID NOs:28 to 31) weresynthesized by arranging them in alternating order of sense chain andantisense chain.

Each of the synthetic DNA fragments (SEQ ID NOs:28 to 31) was added to50 μl of a reaction solution to a final concentration of 0.1 μmol/l, andPCR was carried out in the same manner as in the above-mentioned (2)using 0.5μ mol/l of T3 primer (manufactured by Takara Shuzo), 0.5μ mol/lof T7 primer (manufactured by Takara Shuzo) and 1 unit of KOD polymerase(manufactured by TOYOBO), in accordance with the instructions attachedto the KOD polymerase. The reaction solution was subjected to ethanolprecipitation, and the precipitate was dissolved in sterile water,subjected to an appropriate restriction enzyme treatment and thenconnected to a plasmid pBluescript II SK(−). E. coli DH5α wastransformed using the thus obtained recombinant plasmid DNA solution,each plasmid DNA was prepared from each of the transformants, and thenits nucleotide sequence was analyzed using Big Dye Terminator CycleSequencing Kit (manufactured by Applied Biosystems). As a result, aplasmid pBS/LV0 having the nucleotide sequence of interest was obtained.

Next, the FR amino acid residues designed in this Example (1) wasmodified by preparing a synthetic DNA having mutation and carrying outthe above-mentioned PCR, or by carrying out PCR using a plasmid DNAcontaining a cDNA encoding the LV0 prepared in the above, as thetemplate, and synthetic DNA fragments as primers, and isolating anamplified gene fragment. Gene codons of the amino acid residues aftermodification were prepared in such a manner that they became the genecodons found in the anti-HB-EGF mouse antibody KM3566.

The PCR was carried out by 35 cycles, each cycle consisting of reactionsat 94° C. for 30 seconds, at 55° C. for 30 seconds and at 72° C. for 60seconds, using KOD-plus polymerase (manufactured by TOYOBO). Also, thesynthetic DNA used herein is a product of FASMAC Co., Ltd.

Next, the VL wherein Leu¹⁵Ala¹⁹Ile²¹Pro⁴⁹Leu⁸⁴ of the above LV0 ismodified to Val¹⁵Val¹⁹Met²¹Ser⁴⁹Val⁸⁴ (hereinafter referred to as LV5)was prepared by the following method.

PCR was carried out by adding 0.1 μmol/l of synthetic DNAs having aminoacid mutation (SEQ ID NOs:37 to 40) and 0.4 μmol/l of primers M13RVprimer (manufactured by Takara Shuzo) and M13M4 primer (manufactured byTakara Shuzo) positioning at both termini thereof. The PCR solution waspurified using a Gel extraction kit, 0.8 to 1.5% agarose electrophoresiswas carried out and the intended gene fragment of about 0.45 kbp wasextracted using the Gel extraction kit. Sub-cloning into pBS which hadbeen treated with a specific restriction enzyme SmaI was carried out toobtain a vector pBS/LV5 that contains a gene (SEQ ID NO:41) coding forthe VL amino acid sequence LV5 of anti-HB-EGF humanized antibody.

(4) Construction of Anti-HB-EGF Humanized Antibody Expression Vector

Various anti-HB-EGF humanized antibody expression vectors wereconstructed by inserting a cDNA encoding the HV0 or LV0 obtained in thisExample (2) or (3), or a cDNA encoding a modified product thereof, intoan appropriate position of the vector pKANTEX93 for humanized antibodyexpression described in WO 97/10354.

(5) Stable Expression of Anti-HB-EGF Humanized Antibody and Preparationof Purified Antibody Using an Animal Cell

Stable expression of anti-HB-EGF humanized antibody and purification ofthe antibody from a culture supernatant, using an animal cell, werecarried out in the same manner as the methods described in Example 6(2)and (3). As a result, three anti-HB-EGF humanized antibodies HV0LV0 inwhich the antibody VH, and VL are HV0 and LV0, respectively, HV0LV0 inwhich the antibody VH and VL are HV7 and LV0, respectively, and HV7LV5in which the antibody VH and VL are HV7 and LV5, respectively, wereprepared.

EXAMPLE 10 (1) Measurement of Binding Activity of the Anti-HB-EGFHumanized Antibodies for Human HB-EGF

In order to reaction-kinetically analyze binding activity of theanti-HB-EGF humanized antibodies for human HB-EGF, it was measured inthe same manner as in Example 7(2) using BIACORE T100 (manufactured byBIACORE).

As a result, similar to the results of the anti-HB-EGF chimeric antibodyKM3966 in Example 2(2), the anti-HB-EGF humanized antibodies prepared inExample 9 hardly showed dissociation reaction after binding with humanHB-EGF so that the dissociation rate constant was not able to becalculated.

On the other hand, it was able to calculate the binding rate constant,with the results shown in Table 2. As a result, human HB-EGF bindingactivity of the anti-HB-EGF humanized antibody HV0LV0 in which the CDRsof anti-HB-EGF chimeric antibody KM3966 were simply transplanted intohuman framework was reduced close to about ½ of that of the anti-HB-EGFchimeric antibody KM3966, but the binding activity of the anti-HB-EGFhumanized antibody HV7LV0 in which amino acid modification was added tothe H chain alone and the anti-HB-EGF humanized antibody HV7LV5 in whichamino acid modification was added to the H chain and L chain wasincreased to almost the same level of the anti-HB-EGF chimeric antibodyKM3966.

TABLE 2 Antibody Ka (1/Ms) KM3966 1.87 × 10⁵ HV0LV0 1.04 × 10⁵ HV7LV01.74 × 10⁵ HV7LV5 1.70 × 10⁵

(2) Reactivity of the Anti-HB-EGF Humanized Antibodies for an HB-EGFExpressing Cell

Reactivity of the anti-HB-EGF humanized antibodies for an HB-EGFexpressing cancer cell was measured in the same manner as in Example2(1). A human ovarian cancer cell line MCAS was used as the cell line.The results are shown in FIG. 15. Similar to the case of anti-HB-EGFchimeric antibody KM3966, all of the humanized antibodies reacted withMCAS.

(3) Neutralization Activity of the Anti-HB-EGF Humanized Antibodies

The neutralization activity of the anti-HB-EGF humanized antibodies wasmeasured in the same manner as in Example 2(4).

The results are shown in FIG. 16. When the cell growth at the time ofnot adding HB-EGF was regarded as 1, cell growth was observed by MKN-28due to the exogenous HB-EGF, thus showing an HB-EGF-dependent growth.The anti-HB-EGF humanized antibody HV0LV0 which showed about ½ of thebinding activity of the anti-HB-EGF chimeric antibody KM3966 wasslightly lower in its neutralizing activity than the anti-HB-EGFchimeric antibody KM3966, but the anti-HB-EGF humanized antibodiesHV7LV0 and HV7LV5 having almost the same human HB-EGF binding activityas the anti-HB-EGF chimeric antibody KM3966 showed equivalentneutralizing activity of the anti-HB-EGF chimeric antibody KM3966.

(4) ADCC Activity of the Anti-HB-EGF Humanized Antibodies

The ADCC activity of the anti-HB-EGF humanized antibodies was measuredin the same manner as in Example 2(5).

The results are shown in FIG. 17. ADCC activity of the anti-HB-EGFhumanized antibody HV0LV0 which showed about ½ of the binding activityof the anti-HB-EGF chimeric antibody KM3966 was reduced to about 1/10 incomparison with the anti-HB-EGF chimeric antibody KM3966, but theanti-HB-EGF humanized antibodies HV7LV0 and HV7LV5 having almost thesame as human HB-EGF binding activity of the anti-HB-EGF chimericantibody KM3966 showed equivalent ADCC activity of the anti-HB-EGFchimeric antibody KM3966.

EXAMPLE 11 Analysis on Binding Epitopes of Anti-HB-EGF Antibodies

The following analysis was carried out on the binding epitopes ofanti-HB-EGF antibodies KM3566, KM3579 and KM3966 for human HB-EGF.

(1) Construction of Mutation Type Human HB-EGF Complete LengthGene-Transferred Cell

All of the anti-HB-EGF antibodies KM3566, KM3579, and KM3966 react withhuman HB-EGF, and do not show cross reaction with mouse HB-EGF. Thus,gene-transferred cells which express 10 kinds of mutation type humanHB-EGF complete length proteins (to be referred to as mutant HB-EGFhereinafter) in which each of 10 amino acids in the amino acid sequenceof EGF-like domain of human HB-EGF, which are different from mouseHB-EGF, was substituted with a mouse-derived amino acid wereconstructed, and binding epitopes were analyzed by measuring bindingactivity of anti-HB-EGF antibodies for these. The prepared 10 kinds ofmutant HB-EGF are shown below.

(1) A mutant HB-EGF in which phenylalanine at position 115 from theN-terminal was substituted with tyrosine (hereinafter referred to as“F115Y”)(2) A mutant HB-EGF in which lysine at position 122 from the N-terminalwas substituted with arginine (hereinafter referred to as “K122R”)(3) A mutant HB-EGF in which valine at position 124 from the N-terminalwas substituted with leucine (hereinafter referred to as “V124L”)(4) A mutant HB-EGF in which lysine at position 125 from the N-terminalwas substituted with glutamine (hereinafter referred to as “K125Q”)(5) A mutant HB-EGF in which leucine at position 127 from the N-terminalwas substituted with phenylalanine (hereinafter referred to as “L127F”)(6) A mutant HB-EGF in which alanine at position 129 from the N-terminalwas substituted with threonine (hereinafter referred to as “A129T”)(7) A mutant HB-EGF in which isoleucine at position 133 from theN-terminal was substituted with lysine (hereinafter referred to as“I133K”)(8) A mutant HB-EGF in which histidine at position 135 from theN-terminal was substituted with leucine (hereinafter referred to as“H135L”)(9) A mutant HB-EGF in which glutamic acid at position 141 from theN-terminal was substituted with histidine (hereinafter referred to as“E141H”)(10) A mutant HB-EGF in which serine at position 147 from the N-terminalwas substituted with threonine (hereinafter referred to as “S147T”).

In addition, the following human/mouse chimeric HB-EGF complete lengthgene-transferred cell was constructed as the positive control.

(11) A human/mouse chimeric HB-EGF in which a sequence of positions 1 to49 from N-terminal consists of a mouse HB-EGF-derived sequence and asequence of positions 50 to 208 from N-terminal consists of a humanHB-EGF-derived sequence was prepared. Since all of the EGF-like domainsare human HB-EGF-derived sequences, this HB-EGF was used as the positivecontrol.

The plasmids for transient expression of the above-mentioned mutantHB-EGF and human/mouse chimeric HB-EGF were prepared using the method ofMekada et al. (J. Bio. Chem., 272, 27084-27090 (1997)). Mouse LMTK cell(ATCC CCL-1.3) was cultured using Dulbecco's modified Eagle's mediumsupplemented with 100 unit/ml of penicillin G, 100 μg/ml of streptomycinand 10% bovine serum albumin. Each of the above-mentioned expressionplasmids was transferred into the mouse LMTK cell by the calciumphosphate method, and then the cells were cultured for 48 hours and usedin the following test.

Cell prepared by transferring the vector alone into the mouse LMTK cell(hereinafter referred to as “mock”) was used as the negative control.

(2) Binding Activity Analysis of Anti-HB-EGF Antibody Using MutantHB-EGF Gene-Transferred Cell

Firstly, biotin-labeled anti-HB-EGF antibody (KM3566, KM3579 or KM3966)diluted to 2 μg/ml with a binding buffer (prepared by addingnonessential amino acids, 20 mM HEPES-NaOH (pH 7.2) and 10% fetal bovineserum to Ham's F12) was allowed to react at 4° C. for 2 hours with 1×10⁵cells of the mutant HB-EGF gene-transferred cell, human/mouse chimericHB-EGF gene-transferred cell or mock. After the reaction, the cells werewashed twice with an ice-cooled washing buffer (prepared by adding 0.5mM CaCl₂, 0.5 mM MgCl₂ and 0.1% fetal bovine serum to PBS) and then oncewith PBS(+) (prepared by adding 0.5 mM CaCl₂ and 0.5 mM MgCl₂ to PBS). Aformaldehyde solution diluted to 1.8% with PBS(+) was added to thewashed cells, and the cells were fixed at 4° C. for 20 minutes. Next,the cells were washed once with PBS(+) and then treated with a glycinesolution (0.2 M glycine, 100 mM Tris, pH 8.1) at 4° C. for 20 minutesand subsequently incubated in the washing buffer at 4° C. for 20minutes. Next, the cells were allowed to react with HRP-conjugatedstreptoavidin diluted to 0.1 μg/ml with the binding buffer at 4° C. for20 minutes and washed twice with the washing buffer and twice withPBS(+). By carrying out development of color using a peroxidasedetection kit (ELISA POD Substrate OPD Kit, manufactured by NacalaiTesque), the absorbance at 492 nm was measured and the activity ofcell-bonded HRP was measured.

A value obtained by subtracting the absorbance for mock from theabsorbance for each of the various mutant HB-EGF gene-transferred cellsor human/mouse chimeric HB-EGF gene-transferred cells of the anti-HB-EGFantibody was used as the value A.

Next, in order to analyze expression of the mutant HB-EGF proteinexpressed on the cell membrane of mouse LMTK cell, the absorbance of ananti-HB-EGF rabbit polyclonal antibody which evenly binds to all of themutant HB-EGF (antibody name; H-6, an antibody prepared by immunizing arabbit with a synthetic peptide of positions 54 to 73 from theN-terminal of human HB-EGF, which was crosslinked to Sepharose CL-6B)for the mutant HB-EGF gene-transferred cell, human/mouse chimeric HB-EGFgene-transferred cell and mock was measured by the same method describedin the above. However, the biotin-labeled H-6 antibody was used at anantibody concentration of 10 μg/ml. A value obtained by subtracting theabsorbance for mock from the absorbance for each of the each mutantHB-EGF gene-transferred cell or human/mouse chimeric HB-EGFgene-transferred cell of the H-6 antibody was used as the value B.

In order to correct difference in the expressed amount between thegene-transferred cells, an A/B value was calculated by dividing value Aby value B. The ratio of A/B value for each mutant HB-EGF when A/B valueof anti-HB-EGF antibody for the positive control pRTHGC-6 was regardedas 100% was calculated, and this ratio was used as the relative bindingactivity for each mutant HB-EGF.

The results are shown in FIG. 18. In comparison with the pRTHGC-6, theanti-HB-EGF monoclonal antibody KM3566 hardly bound to 1133K, H135L andS147T. Accordingly, it was found that the anti-HB-EGF monoclonalantibody KM3566 recognizes an epitope comprising amino acids of I atposition 133, H at position 135 and S at position 147. In addition,similar to the case of anti-HB-EGF monoclonal antibody KM3566, theanti-HB-EGF monoclonal antibody KM3966 having the same antibody variableregion also hardly bound to 1133K and H135L in comparison with thepRTHGC-6, and the binding activity for S147T was decreased to about ⅓.Thus, it was found that the anti-HB-EGF monoclonal antibody KM3966recognizes an epitope comprising amino acids of I at position 133, H atposition 135 and S at position 147, similar to the case of anti-HB-EGFmonoclonal antibody KM3566.

In comparison with the pRTHGC-6, the anti-HB-EGF monoclonal antibodyKM3579 did not bind only to E141H, and showed a binding activity ofequivalent to pRTHGC-6 for all of the other mutant HB-EGF. Accordingly,it was found that the anti-HB-EGF monoclonal antibody KM3579 recognizesan epitope comprising the amino acid of E at position 141.

Based on the above results, it was found that the anti-HB-EGF monoclonalantibody KM3566 and anti-HB-EGF monoclonal antibody KM3966, and theanti-HB-EGF monoclonal antibody KM3579 recognize different epitopes ofHB-EGF.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skill in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

This application is based on Japanese patent application No. 2007-315068filed Dec. 5, 2007, the entire contents of which are incorporatedhereinto by reference. All references cited herein are incorporated intheir entirety.

INDUSTRIAL APPLICABILITY

The present invention provides a monoclonal antibody or an antibodyfragment thereof which binds to a cell membrane-bound HB-EGF, a membranetype HB-EGF and a secretory HB-EGF.

FREE TEXT OF SEQUENCE LISTING

SEQ ID NO:18—Description of artificial sequence: Amplification primer ofKM3966HSEQ ID NO:19—Description of artificial sequence: Amplification primer ofKM3966HSEQ ID NO:20—Description of artificial sequence: Amplification primer ofKM3966LSEQ ID NO:21—Description of artificial sequence: Amplification primer ofKM3966LSEQ ID NO:22—Description of artificial sequence: Amino acid sequence ofHV0 of the anti-HB-EGF humanized antibodySEQ ID NO:23—Description of artificial sequence: Amino acid sequence ofLV0 of the anti-HB-EGF humanized antibodySEQ ID NO:24—Description of artificial sequence: Amplification primer ofHV0SEQ ID NO:25—Description of artificial sequence: Amplification primer ofHV0SEQ ID NO:26—Description of artificial sequence: Amplification primer ofHV0SEQ ID NO:27—Description of artificial sequence: Amplification primer ofHV0SEQ ID NO:28—Description of artificial sequence: Amplification primer ofLV0SEQ ID NO:29—Description of artificial sequence: Amplification primer ofLV0SEQ ID NO:30—Description of artificial sequence: Amplification primer ofLV0SEQ ID NO:31—Description of artificial sequence: Amplification primer ofLV0SEQ ID NO:32—Description of artificial sequence: Amplification primer ofHV7SEQ ID NO:33—Description of artificial sequence: Amplification primer ofHV7SEQ ID NO:34—Description of artificial sequence: Amplification primer ofHV7SEQ ID NO:35—Description of artificial sequence: Amplification primer ofHV7SEQ ID NO:36—Description of artificial sequence: Nucleotide sequence ofHV7SEQ ID NO:37—Description of artificial sequence: Amplification primer ofLV5SEQ ID NO:38—Description of artificial sequence: Amplification primer ofLV5SEQ ID NO:39—Description of artificial sequence: Amplification primer ofLV5SEQ ID NO:40—Description of artificial sequence: Amplification primer ofLV5SEQ ID NO:41—Description of artificial sequence: Nucleotide sequence ofLV5SEQ ID NO:42—Description of artificial sequence: Amino acid sequence ofHV7 of the anti-HB-EGF humanized antibodySEQ ID NO:43—Description of artificial sequence: Amino acid sequence ofLV5 of the anti-HB-EGF humanized antibody

1-17. (canceled)
 18. An isolated antibody or fragment thereof, whereinsaid antibody or said fragment comprises at least one heavy chainvariable region and at least one light chain variable region, whereinsaid heavy chain variable region comprises the amino acid sequence ofSEQ ID NO:22, and wherein said light chain variable region comprises theamino acid sequence of SEQ ID NO:43.
 19. An isolated antibody orfragment thereof, wherein said antibody or said fragment comprises atleast one heavy chain variable region and at least one light chainvariable region, wherein said heavy chain variable region comprises theamino acid sequence of SEQ ID NO:42, and wherein said light chainvariable region comprises the amino acid sequence of SEQ ID NO:23. 20.An isolated antibody fragment thereof, wherein said antibody or saidfragment comprises at least one heavy chain variable region and at leastone light chain variable region, wherein said heavy chain variableregion, wherein said heavy chain variable region comprises the aminoacid sequence of SEQ ID NO:42, and wherein said light chain variableregion comprises the amino acid sequence of SEQ ID NO:43.
 21. Theantibody fragment according to any one of claims 18 to 20, 29 and 30,which is selected from Fab, Fab′, F(ab′)₂, a single chain antibody(scFv), a dimerized V region (diabody), a disulfide stabilized V region(dsFv), and a peptide comprising six CDRs.
 22. A DNA encoding theantibody or the antibody fragment thereof according to any one of claims18 to 20, 29 and
 30. 23. A recombinant vector comprising the DNAaccording to claim
 22. 24. A transformant obtainable by introducing therecombinant vector according to claim 23 into a host cell.
 25. A processfor producing the antibody or the antibody fragment thereof according toany one of claims 18 to 20, 29 and 30, which comprises culturing thetransformant according to claim 24 in a medium to form and accumulatethe antibody or the antibody fragment thereof according to any one ofclaims 18 to 20, 29 and 30 in the culture, and recovering the antibodyor the antibody fragment from the culture.
 26. A pharmaceuticalcomposition comprising the antibody or the antibody fragment thereofaccording to any one of claims 18 to 20, 29 and 30 as active ingredient.27. An agent for treating a disease relating to HB-EGF, comprising theantibody or the antibody fragment thereof according to any one of claims18 to 20, 29 and 30 as an active ingredient.
 28. The agent according toclaim 27, wherein the disease relating to HB-EGF is cancer.
 29. Anisolated antibody or fragment thereof, wherein said antibody or saidfragment comprises at least one heavy chain variable region and at leastone light chain variable region, wherein said heavy chain variableregion comprises three complementarity—determining regions comprisingthe amino acid sequences of SEQ ID NOS:12, 13 and 14; and wherein saidlight chain variable region comprises the amino acid sequence of SEQ IDNO:43.
 30. An isolated antibody or fragment thereof, wherein saidantibody or said fragment comprises at least one heavy chain variableregion and at least one light chain variable region, wherein said heavychain variable region comprises the amino acid sequence of SEQ ID NO:42;and wherein said light chain variable region comprises at least threecomplementarity-determining regions comprising the amino acid sequencesof SEQ ID NOS:15, 16 and 17.